METAP2 inhibitors and methods of treating obesity

ABSTRACT

The present invention relates to modified or polymer conjugated MetAP2 inhibitors. The present invention also relates to methods of preventing, inducing, causing or increasing weight loss, treating obesity and/or treating metabolic syndrome utilizing the modified or polymer conjugated MetAP2 inhibitors. The present invention also relates to methods of improving insulin sensitivity and glycemic control, reducing insulin levels and/or improving leptin sensitivity utilizing the modified or polymer conjugated MetAP2 inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/851,668, filed Sep. 11, 2015, which is a continuation of U.S.application Ser. No. 14/248,787, filed Apr. 9, 2014, now U.S. Pat. No.9,173,956, issued Nov. 3, 2015 which claims the benefit of U.S.Provisional Application No. 61/810,468, filed Apr. 10, 2013 and U.S.Provisional Application No. 61/925,918, filed Jan. 10, 2014. Thecontents of each of these applications are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Obesity is a chronic disease and a major health concern in modernsociety. According to the Centers for Disease Control (CDC), the UnitedStates is in the midst of an epidemic of obesity. In the U.S., about 65%of adults are overweight, 30% of adults are obese, with more than 5million adults classified as morbidly obese. Ten million more are nearthat mark and may be at risk for obesity-related health problems. Theproblem is increasing; obesity in children and adolescents increasedtwo-fold in the last two decades.

Existing therapies for obesity include standard diets and exercise, verylow calorie diets, behavioral therapy, pharmacotherapy involvingappetite suppressants, thermogenic drugs, food absorption inhibitors,mechanical devices such as jaw wiring, waist cords and balloons, andsurgery. However, these existing therapies are not very effective.Adherence to energy restriction diets is problematic and generallyunsuccessful and medical therapies have only modest efficacy forlong-term weight management. In most cases, toxicity and side effectshave hampered the development of potential weight loss drug candidates.Metabolic syndrome (Sutherland, et al., Metabolic Syndrome and RelatedDisorders 2:82-104 (2004); Esposito, et al., Nutr. Metab. Cardiovasc.Dis. 14:228-232 (2004)), relates to obesity and is characterized by agroup of metabolic risk factors including: 1) abdominal obesity(excessive fat tissue in and around the abdomen); 2) atherogenicdyslipidemia (high triglycerides; low HDL cholesterol and high LDLcholesterol); 3) elevated blood pressure; 4) insulin resistance orglucose intolerance; 5) a prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in the blood); and 6) aproinflammatory state (e.g., elevated CRP in the blood). Metabolicsyndrome has become increasingly common in developed countries and isclosely associated with risk of coronary heart disease (Malik, et al.,Circulation 110:1245-1250 (2004); Irabarren, et al., J. Am. Coll.Cardiol. 48:1800-1807 (2006)).

Cardiometabolic syndrome includes obesity-related metabolic disordersand atherosclerosis. Cardiometabolic disorders also promote arterial andvalvular calcification which may lead to devastating clinicalcomplications: acute myocardial infarction and aortic stenosis. Inaddition, diabetes causes chronic kidney disease that also leads tocardiovascular ectopic calcification and acute myocardial infarction.Collectively, several major components of the cardiometabolic syndrome,developed via interrelated mechanisms, enhance each other through localor systemic inflammation. Further, lack of patient adherence toprescribed medications poses a tremendous challenge to the globalhealthcare community. In the US alone, avoidable medical spending wasestimated at $300 billion in 2009. With blockbuster expiries, dryingpipelines and increasing cost-containment by payers, bridging theadherence gap is a “must do” for pharmaceutical companies.

Accordingly, new compounds and methods for causing, inducing and/orincreasing weight loss and treating obesity and metabolic syndrome areneeded. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention provides methods of inducing or causing weightloss in a subject in need thereof comprising administering at least onecompound of the present invention in a therapeutically effective amountto the subject to induce or cause weight loss. In certain embodiments,the subject is overweight or obese. In certain embodiments, inducing orcausing weight loss is increasing weight loss.

The present invention also provides methods of treating obesity,metabolic syndrome and/or related co-morbidities in a subject in needthereof comprising administering at least one compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof, in a therapeutically effective amount on areasonable schedule to the subject to treat or ameliorate these diseasesand conditions.

The present invention also provides methods of improving insulinsensitivity and glycemic control, reducing insulin levels and/orimprovements in leptin sensitivity in a subject in need thereofcomprising administering at least one compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, in a therapeutically effective amount on areasonable schedule to the subject to treat or ameliorate these diseasesand conditions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing body weight change over time followingadministration of compounds of the present invention.

FIG. 2 is a graph showing average weekly food consumption followingadministration of compounds of the present invention.

FIG. 3 is a graph showing the relationship between total fat weight andbody weight following administration of compounds of the presentinvention.

FIG. 4 is a graph showing blood glucose levels following administrationof a glucose challenge during the treatment period of a murine studyusing compounds of the present invention.

FIG. 5 is a graph showing body weight change over time followingadministration of various dosages of a compound of the present inventionon a q4d dosing schedule.

FIG. 6 is a graph showing changes in total cholesterol, triglycerides,HDL cholesterol and LDL cholesterol at the conclusion of a 32 day studyas a function of dose level using compounds of the present invention.

FIG. 7 is a graph showing body weight change over time followingadministration of various dosages of a compound of the present inventionto rats on a q7d dosing schedule.

FIG. 8 is a graph showing the change in body weight of rats treated witha single dose of various test agents.

FIG. 9 is a graph showing the plasma concentration of a compound of thepresent invention over time based on administration of two differentcompounds.

FIG. 10 is a graph showing body weight change over time followingadministration of various compounds of the present invention.

FIG. 11 is a graph showing body weight changes in male Levin rats on ahigh fat diet following administration of various compounds of thepresent invention.

FIG. 12 is a graph showing fumagillol exposure versus weight loss in DIOrats following administration of various compounds of the presentinvention.

FIG. 13 is a graph showing changes in insulin levels in male Levin DIOrats on a high fat diet following administration of various compounds ofthe present invention.

FIG. 14 is a graph showing insulin levels during an oral glucosechallenge (OGTT) following administration of various compounds of thepresent invention.

FIG. 15 is a graph showing reductions in blood glucose in DIO ratsduring an oral glucose challenge (OGTT) following administration ofvarious compounds of the present invention.

FIG. 16 is a graph showing HOMA-ir product during an OGTT in DIO ratsfollowing administration of various compounds of the present invention.

FIG. 17 is a graph showing weekly food consumption followingadministration of various compounds of the present invention.

FIG. 18 is a graph showing changes in leptin levels from baselinefollowing administration of various compounds of the present invention.

FIG. 19 is a graph showing body weight changes in DIO mice followingadministration of various compounds of the present invention on Q4D andQ8D schedules.

FIG. 20 is a graph showing weight loss following administration ofCKD-732 on a Q2D and Q4D Schedule.

FIG. 21 is a graph showing the reductions in food intake followingadministration of various compounds of the present invention.

FIG. 22 is a graph showing significantly reduced insulin levels duringan ipGTT in male C57Bl6 mice kept on a high fat diet for 25 weeksfollowing administration of various compounds of the present invention.

FIG. 23 is a graph showing insulin AUC during a glucose challenge inmale DIO mice following administration of various compounds of thepresent invention.

FIG. 24 is a graph showing blood glucose levels in male C57Bl6 mice on ahigh fat diet following administration of various compounds of thepresent invention.

FIG. 25 is a graph showing HOMA product during an ipGTT in C57Bl6 Miceon a high fat diet following administration of various compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the Present Invention

The present invention provides drug conjugate compositions including anactive moiety modified, a conjugate moiety, and a cleavable linker,wherein cleavage of the linker occurs substantially in a target tissueto produce a modified active moiety having reduced efflux from targettissue compared to the unmodified active moiety. The present inventionalso provides compositions including a modified active moiety.

The conjugate moiety used depends on the physicochemical properties ofboth the conjugate moiety and the active moiety, in addition tobiological requirements, e.g., pharmacokinetic and pharmacodynamicproperties of the active moiety and knowledge of the disease state. Oneof skill in the art will be able to select an appropriate conjugatemoiety based upon the above considerations. The conjugate moiety may beused to deliver small molecule active moieties or larger molecule activemoieties, such as proteins, peptides, or oligonucleotides.

The conjugate moiety improves the delivery of an active moiety totarget. The conjugate moiety is chosen to maximize bioavailability ofthe active moiety, optimize onset, duration, and rate of delivery of theactive moiety, and maintain the concentration of an active moiety in atarget tissue within a therapeutic range as long as required foreffective treatment. The conjugate moiety may also assist in minimizingadverse side effects of an active moiety. Thus the conjugate moietyprolongs pharmacological activity of an active moiety, stabilizes labileactive moieties from chemical and proteolytic degradation, minimizesside effects, increases solubility, and targets the active moiety tospecific cells or tissues.

Other properties of the conjugate moiety to be considered are that theconjugate moiety is minimally or non-immunogenic and non-toxic. Themolecular weight of the conjugate moiety should be sufficiently large toavoid rapid elimination via kidney ultrafiltration and low enough toprevent undesirable accumulation within the body. In certainembodiments, the conjugate moiety is hydrophilic and is biodegradable.Conjugate moieties that are non-biodegradable are also suitable withcompositions and methods of the invention. The conjugate moiety shouldbe able to carry the required amount of active moiety and protectagainst premature metabolism of the active moiety in transit to thetarget tissue.

Exemplary conjugates include all forms of polymers, synthetic polymersas well as natural product related polymers including peptides,polysaccharides, polynucleic acids, antibodies and aptamers. Inpreferable embodiments, the conjugate is a synthetic polymer. Exemplarypolymers of the invention have been described in U.S. Pat. No. 4,997,878to Bock et al, U.S. Pat. No. 5,037,883 to Kopecek et al. U.S. Pat. No.5,258,453 to Kopecek et al., U.S. Pat. No. 6,464,850 to Zhang et al.,U.S. Pat. No. 6,803,438 to Brocchini et al., each of which isincorporated by reference in its entirety. Additional exemplary polymershave been described in Subr et al., J Controlled Release, 18, 123-132(1992). In some embodiments, the method of synthesis of the polymer maylead to the coupling of two or more polymer chains and may increase theweight average molecular weight of the polymer conjugate. It is furtherrecognized that if this coupling occurs, the linkages will bebiodegradable.

The active moiety may be any compound or molecule that produces atherapeutic effect in a subject. In certain embodiments, the compound ormolecule has a molecular weight of 2000 Daltons or less, 1500 Daltons orless, 1000 Daltons or less, 500 Daltons or less, or 250 Daltons or less.In certain embodiments, the compound or molecule is a MetAP2 inhibitor.In certain embodiments, the compound or molecule is fumagillin,fumagillol, or an analog, derivative, salt or ester thereof. Thecompound or molecule chosen will depend on the condition or disease tobe treated. In certain embodiments, two or more active moieties may beused. In certain embodiments an active moiety and an inactive “capping”moiety may be used. In certain embodiments, the condition to be treatedis obesity. In compositions of the invention, the conjugate moiety isjoined to the active moiety via a linker. Any linker structure known inthe art may be used to join the modified active moiety to the conjugatemoiety. The linker used will depend on the physiological conditions ofthe target tissue, the properties of the active moiety that are beingoptimized, and the cleavage mechanism. D'Souza et al. review varioustypes of linkers including linkers that operate via proteolytic cleavage“Release from Polymeric Prodrugs: Linkages and Their Degradation” J.Pharm. Sci., 93, 1962-1979 (2004). Blencoe et al. describe a variety ofself-immolative linkers, “Self-immolative linkers in polymeric deliverysystems” Polym. Chem. 2, 773-790 (2011). Ducry et al. review linkers inBioconj. Chem. 21, 5-13 (2010) “Antibody-Drug Conjugates: LinkingCytotoxic Payloads to Monoclonal Antibodies”. Peptide linkers suitablefor cleavage by matrix metalloproteins (MMPs) are described in Chau etal. “Antitumor efficacy of a novel polymer-peptide-drug conjugate inhuman tumor xenograft models” Int. J. Cancer 118, 1519-1526 (2006) andChau et al. U.S. patent publication number 2004/0116348. Other linkerchemistries suitable with compositions of the invention are shown inShiose et al. Biol. Pharm. Bull. 30(12) 2365-2370 (2007); Shiose et al.Bioconjugate Chem. 20(1) 60-70 (2009); Senter, U.S. Pat. No. 7,553,816;De Groot, U.S. Pat. No. 7,223,837; King, U.S. Pat. No. 6,759,509;Susaki, U.S. Pat. No. 6,835,807; Susaki U.S. Pat. No. 6,436,912; andGemeinhart U.S. Pat. No. 7,943,569.

In certain embodiments, the linker is a peptide linker. Exemplarypeptide linkers are described in U.S. Pat. No. 6,835,807 to Susaki etal., U.S. Pat. No. 6,291,671 to Inoue et al., U.S. Pat. No. 6,811,996 toInoue et al., U.S. Pat. No. 7,041,818 to Susaki et al., U.S. Pat. No.7,091,186 to Senter et al., U.S. Pat. No. 7,553,816 to Senter et al.each of which is incorporated by reference in its entirety. Additionalexemplary peptides and their cleavage have been described in Shiose etal. Biol. Pharm. Bull. 30(12) 2365-2370 (2007) and Shiose et al.Bioconjugate Chem. 20(1) 60-70 (2009). Peptide linkers suitable forcleavage by matrix metalloproteins (MMPs) are described in Chau et al.“Antitumor efficacy of a novel polymer-peptide-drug conjugate in humantumor xenograft models” Int. J. Cancer 118, 1519-1526 (2006) and Chau etal. U.S. patent publication number 2004/0116348.

The linker may be cleaved by any mechanism known in the art. Forexample, the linkers may be designed for proteolytic cleavage orintracellular proteolytic cleavage. In certain embodiments, the linkeris designed such that there is no cleavage of the linker in plasma orthere is a very low rate of cleavage in the plasma. Exemplary linkerstructures are described in further detail below.

In certain embodiments, the linker has a structure such that it is to bepreferentially cleaved in disease tissue. Since most hydrolases exist inboth normal and diseased tissue, the linker should be cleaved by ahydrolase that is more active in disease tissue and/or more prevalent indisease tissue. For example, tumors have generally upregulated metabolicrates and in particular over express proteases including the cathepsins.The upregulation and role of proteases in cancer is described by Masonet al. Trends in Cell Biology 21, 228-237 (2011).

In certain embodiments, the class of active moieties that are modifiedare moieties that irreversibly bind to their targets, i.e., afterrelease from the conjugate the active moiety covalently binds to thebiochemical target. Once bound, the active moiety cannot diffuse or betransported out of the cell. For targeting to occur in the case ofirreversible binding, the rate of small molecule binding to target,k_(rev1), should be significant relative to the rate of small moleculeefflux, k_(sm-1). If the rate of efflux is high relative to smallmolecule binding, small molecule equilibrium will be established betweenthe plasma and the intracellular compartment and there will be noadvantage to intracellular delivery relative to extracellular delivery.

In other embodiments, the class of active moieties that are modified aremoieties that reversibly bind to their targets. For targeting to occurin the case of reversible binding, the equilibrium constant for smallmolecule binding to target K=k_(rev1)/k_(rev-1) should be large and the“on-rate”, k_(rev1), should be large relative to the rate of smallmolecule efflux, k_(sm-1). If the rate of efflux is high relative tosmall molecule binding, small molecule equilibrium will be establishedbetween the plasma and the intracellular compartment and there will beno advantage to intracellular delivery relative to extracellulardelivery. Such a relationship is described schematically below, where:[PC]=concentration of polymer conjugate; [SM]=concentration of releasedsmall molecule; plasma=plasma concentration; icell=intracellularconcentration; icell-target=small molecule reversibly bound tointracellular target; and inactive=inactive metabolite of smallmolecule. In certain embodiments, when k_(rev-1)=zero, the moietyirreversibly binds to its target.

In other embodiments, the class of active moieties that are modified aremoieties that have very high equilibrium constants and high “on-rates”relative to efflux. In other embodiments, the class of active moietiesthat are modified are moieties that undergo intracellular metabolism ata high rate relative to efflux.

In certain embodiments, modifications to the active moiety areaccomplished by using a linker having a structure such that uponcleavage, a fragment of the linker remains attached to the activemoiety. That fragment may change any of the molecular weight,hydrophobicity, polar surface area, or charge of the active moiety,thereby producing a modified active moiety having reduced efflux from atarget cell compared to the unmodified active moiety. For example,coupling MetAP2 inhibitory active moieties via the linkers describedherein provide conjugates in which upon cleavage of the linker, producean active moiety having a fragment of the linker attached thereto(modified active moiety). The modified active moieties described hereinmay have reduced efflux from a cell compared to the unmodified activemoieties, resulting in modified active moieties with superior efficacyto the parent small molecules and superior efficacy to the parent smallmolecules and superior pharmacokinetic profiles.

The present invention provides conjugates with linkers having thestructure:

wherein, independently for each occurrence, R₄ is H or C₁-C₆ alkyl; R₅is H or C₁-C₆ alkyl; R₆ is C₂-C₆ hydroxyalkyl; Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L or—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine, alanine,or H₂N(CH₂)mCO₂H, wherein m is 2, 3, 4 or 5; AA₂ is a bond, or alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine;AA₃ is a bond, or alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, or tyrosine; AA₄ is a bond, or alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine; AA₅ is abond, or glycine, valine, tyrosine, tryptophan, phenylalanine,methionine, leucine, isoleucine, or asparagine; AA₆ is a bond, oralanine, asparagine, citrulline, glutamine, glycine, leucine,methionine, phenylalanine, serine, threonine, tryptophan, tyrosine,valine, or H₂N(CH₂)mCO₂H, wherein m is 2, 3, 4 or 5; L is —OH,—O-succinimide, —O-sulfosuccinimide, alkoxy, aryloxy, acyloxy, aroyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, —NH₂, —NH(C₂-C₆ hydroxyalkyl),halide or perfluoroalkyloxy; Q is NR, O, or S; X isM-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V; M is a bond, or C(O); J is a bond,or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl, heteroaryl, NR, O, or S; Yis NR, O, or S; R is H or alkyl; V is a bond or

R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with Y formsa heterocyclic ring; R¹⁰ is amido or a bond; R¹¹ is H or alkyl; W is aMetAP2 inhibitor moiety or alkyl; x is in the range of 1 to about 450; yis in the range of 1 to about 30; n is in the range of 1 to about 50; pis 0 to 20; q is 2 or 3; and r is 1, 2, 3, 4, 5, or 6.

In certain embodiments, R₄ is C₁-C₆ alkyl. In certain embodiments, R₄ ismethyl. In certain embodiments, R₅ is C₁-C₆ alkyl. In certainembodiments, R₅ is methyl. In certain embodiments, R₆ is 2-hydroxyethyl,2-hydroxypropyl or 3-hydroxypropyl. In certain embodiments, R₆ is2-hydroxypropyl.

In certain embodiments, the compound has a molecular weight of less thanabout 60 kDa. In other embodiments, the molecular weight is less thanabout 45 kDa. In other embodiments, the molecular weight is less thanabout 35 kDa.

In certain embodiments, the ratio of x to y is in the range of about30:1 to about 3:1. In other embodiments, the ratio of x to y is in therange of about 19:2 to about 7:2. In certain embodiments, the ratio of xto y is in the range of about 9:1 to about 4:1. In certain embodiments,the ratio of x to y is about 11:1. In certain embodiments, the ratio ofx to y is about 9:1. In certain embodiments, the ratio of x to y isabout 4:1.

In certain embodiments, Z is —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L. Incertain embodiments, L is methoxy, ethoxy, pentafluorophenyloxy,phenyloxy, acetoxy, fluoride, chloride, methoxycarbonyloxy;ethoxycarbonyloxy, phenyloxycarbonyloxy, 4-nitrophenyloxy,trifluoromethoxy, pentafluoroethoxy, or trifluoroethoxy. In certainembodiments, L is 4-nitrophenyloxy.

In certain embodiments, Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W. In certain embodiments,AA₁ is glycine. In certain embodiments, AA₂ is glycine. In certainembodiments, AA₃ is glycine. In certain embodiments, AA₄ is glycine orphenylalanine. In certain embodiments, AA₅ is leucine, phenylalanine,valine or tyrosine. In certain embodiments, AA₆ is asparagine,citrulline, glutamine, glycine, leucine, methionine, threonine ortyrosine. In certain embodiments, AA₅-AA₆ is Leu-Cit, Leu-Gln, Leu-Gly,Leu-Leu, Leu-Met, Leu-Thr, Phe-Cit, Phe-Gln, Phe-Leu, Phe-Met, Phe-Thr,Val-Asn, Val-Cit, Val-Gln, Val-Leu, Val-Met, Val-Thr, Tyr-Cit, Tyr-Leu,or Tyr-Met. In certain embodiments, AA₁, AA₃ and AA₅ are glycine,valine, tyrosine, tryptophan, phenylalanine, methionine, leucine,isoleucine, or asparagine. In certain embodiments, AA₂, AA₄ and AA₆ areglycine, asparagine, citrulline, glutamine, glycine, leucine,methionine, phenylalanine, threonine or tyrosine. In certainembodiments, AA₂ is a bond; and AA₃ is a bond. In certain embodiments,AA₁ is glycine; AA₄ is phenylalanine; AA₅ is leucine; and AA₆ isglycine.

In certain embodiments, W is

wherein R₂ is —OH or methoxy; and R₃ is H, —OH or methoxy.

In certain embodiments, W is

In certain embodiments, W is

In certain embodiments, Q is NR. In other embodiments, Q is S.

In certain embodiments, J is NR. In other embodiments, J is((CH₂)_(q)Q)_(r). In other embodiments, J is C₅-C₈ cycloalkyl. Incertain embodiments, J is aryl.

In certain embodiments, Y is NR. In other embodiments, Y is S.

In certain embodiments, -Q-X—Y— is

V is:

or a bond; R¹² is H or Me; or R¹² taken together with R¹⁴ forms apiperidine ring; R¹¹ is H or Me; and R¹³ taken together with R¹² forms apiperidine ring.

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, -Q-X—Y— is

In certain embodiments, R₄ and R₅ are methyl; R₆ is 2-hydroxypropyl; Zis —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ isa bond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine; AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, R₄ and R₅ are methyl; R₆ is 2-hydroxypropyl; Zis —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ isa bond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine; AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, R₄ and R₅ are methyl; R₆ is 2-hydroxypropyl; Zis —NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine; AA₂ isa bond; AA₃ is a bond; AA₄ is phenylalanine; AA₅ is leucine; AA₆ isglycine; -Q-X—Y— is

and W is

In certain embodiments, -Q-X—Y— is a self-immolating linker thatreleases the MetAP2 inhibitor in the form of a carbamate derivative, asshown in the scheme below:

Another aspect of the present invention provides conjugates with linkershaving the structure: Z-Q-X—Y—C(O)—W; wherein, independently for eachoccurrence, Z is H₂N-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)— or H; AA₂ is a bond, oralanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine;AA₃ is a bond, or alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, or tyrosine; AA₄ is a bond, or alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine; AA₅ is abond, alanine, cysteine, glycine, isoleucine, leucine, methionine,phenylalanine, valine, tryptophan, or; AA₆ is alanine, asparagine,citrulline, glutamine, glycine, leucine, methionine, phenylalanine,serine, threonine, tryptophan, tyrosine, valine or H₂N(CH₂)mCO₂H,wherein m is 2, 3, 4 or 5; Q is NR, O, or S; X isM-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V; M is a bond, or C(O); J is a bond,or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl, heteroaryl, NR, O, or S; Yis NR, O, or S; R is H or alkyl; V is a bond or

R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with Y formsa heterocyclic ring; R¹⁰ is amido or a bond; R¹¹ is H or alkyl; W is aMetAP2 inhibitor moiety; p is 0 to 20; q is 2 or 3; and r is 1, 2, 3, 4,5, or 6.

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—. In certain embodiments,AA₅ is alanine, cysteine, glycine, isoleucine, leucine, methionine,phenylalanine, valine, tryptophan, or tyrosine and AA₆ is glycine. Incertain embodiments, AA₅ is leucine and AA₆ is glycine. In certainembodiments, AA₅ is valine and AA₆ is glycine. In certain embodiments,AA₅ is phenylalanine and AA₆ is glycine. In certain embodiments, AA₅ isglycine and AA₆ is glycine. In certain embodiments, AA₅ is not valine.

In other embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—. In certainembodiments, AA₅ is alanine, cysteine, glycine, isoleucine, leucine,methionine, phenylalanine, valine, tryptophan, or tyrosine and each ofAA₃, AA₄, or AA₆ is glycine. In certain embodiments, AA₅ is leucine andeach of AA₃, AA₄, or AA₆ is glycine. In certain embodiments, AA₅ isvaline and each of AA₃, AA₄, or AA₆ is glycine. In certain embodiments,AA₅ is phenylalanine and each of AA₃, AA₄, or AA₆ is glycine. In certainembodiments, AA₃ is glycine, AA₄ is phenylalanine, AA₅ is leucine andAA₆ is glycine. In certain embodiments, each of AA₃, AA₄, AA₅ and AA₆ isglycine. In certain embodiments, AA₅ is not valine.

In certain embodiments, Z is H. In other embodiments, Z isH₂N-AA₆-C(O)—. In certain embodiments, AA₆ is glycine.

In certain embodiments, Q is NR. In certain embodiments, M is a bond. Incertain embodiments, J is a bond. In certain embodiments, Y is NR.

In certain embodiments, W is:

wherein R₂ is —OH or methoxy; and R₃ is H, —OH or methoxy.

In certain embodiments, W is

In certain embodiments, W is

In certain embodiments, -Q-X—Y— is

V is:

or a bond; R¹² is H or Me; or R¹² taken together with R¹⁴ forms apiperidine ring; R¹¹ is H or Me; and R¹³ taken together with R¹² forms apiperidine ring.

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is leucine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is valine and AA₆ isglycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is phenylalanine andAA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is glycine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is leucineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is valineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ isphenylalanine and each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₃ is glycine,AA₄ is phenylalanine, AA₅ is leucine and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; each of AA₃,AA₄, AA₅ and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is leucine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is valine and AA₆ isglycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is phenylalanine andAA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is glycine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is leucineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is valineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ isphenylalanine and each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₃ is glycine,AA₄ is phenylalanine, AA₅ is leucine and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; each of AA₃,AA₄, AA₅ and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is leucine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is valine and AA₆ isglycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is phenylalanine andAA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₅-AA₆-C(O)—; AA₅ is glycine and AA₆is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is leucineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ is valineand each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₅ isphenylalanine and each of AA₃, AA₄, or AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; AA₃ is glycine,AA₄ is phenylalanine, AA₅ is leucine and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₃-AA₄-AA₅-AA₆-C(O)—; each of AA₃,AA₄, AA₅ and AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H₂N-AA₆-C(O)—; AA₆ is glycine; Q-X—Y is

and W is

In certain embodiments, Z is H; Q-X—Y is

and W is

Other active moieties that may be modified to be used in conjugates ofthe invention include the following structures:

In certain embodiments, the active moiety is an anti-obesity compound.In other embodiments, the active moiety is a molecule that inhibitsmethionine aminopeptidase-2 (MetAP2), such as fumagillin, fumagillol, oran analog, derivative, salt or ester thereof. Further exemplary MetAP2inhibitors have been described in U.S. Pat. No. 6,242,494 to Craig etal, U.S. Pat. No. 6,063,812 to Hong et al., U.S. Pat. No. 6,887,863 toCraig et al., U.S. Pat. No. 7,030,262 to BaMaung et al., U.S. Pat. No.7,491,718 to Comess et al., each of which is incorporated by referencein its entirety. Additional exemplary MetAP2 inhibitors have beendescribed in Wang et al. “Correlation of tumor growth suppression andmethionine aminopeptidase-2 activity blockade using an orally activeinhibitor,” PNAS 105(6) 1838-1843 (2008); Lee at al. “Design, Synthesis,and Antiangiogenic Effects of a Series of Potent Novel FumagillinAnalogues,” Chem. Pharm. Bull. 55(7) 1024-1029 (2007); Jeong et al.“Total synthesis and antiangiogenic activity of cyclopentane analoguesof fumagillol,” Bioorganic and Medicinal Chemistry Letters 15, 3580-3583(2005); Arico-Muendel et al. “Carbamate Analogues of Fumagillin asPotent, Targeted Inhibitors of Methionine Aminopeptidase-2,” J. Med.Chem. 52, 8047-8056 (2009); and International Publication No. WO2010/003475 to Heinrich et al.

Fumagillin is a small molecule which has been used as an antimicrobialand antiprotozoal agent. Its physiochemical properties and method ofproduction are well known (See U.S. Pat. No. 2,803,586 and Turner, J. R.et al., The Stereochemistry of Fumagillin, Proc. Natl. Acad. Sci. 48,733-735 (1962)). The fermentation product, fumagillin, may be hydrolyzedto yield the alcohol fumagillol which in turn may be converted intovarious derivatives including carbamoylfumagillol, MW 325. The synthesisand preparation of carbamoylfumagillol and some small moleculederivatives are described in U.S. Pat. No. 5,166,172.

Fumagillin and related compounds are believed to exert their biologicaleffects through the inhibition of MetAP2. This enzyme removes N-terminalmethionine from nascent cellular proteins. (See Tucker, L. A., et al.“Ectopic Expression of Methionine Aminopeptidase-2 Causes CellTransformation and Stimulates Proliferation”, Oncogene 27, 3967 (2008).)

Carbamoylfumagillol and derivatives as well as other inhibitors ofMetAP2 have shown therapeutic benefits in preclinical and clinicalstudies. These compounds inhibit cell proliferation and angiogenesis asdescribed in U.S. Pat. No. 5,166,172. Fumagillin analogs or derivatives,such as CKD-732 and PI-2458, are well studied in various systems asdescribed in detail in Bernier et al., “Fumagillin class inhibitors ofmethionine aminopeptidase-2” Drugs of the Future 30(5): 497-508, 2005.

The anti-obesity effects of fumagillin and its analogs are well-known.Rupnick et al. “Adipose tissue mass can be regulated through thevasculature” PNAS 99, 10730-10735, 2002 describes weight loss in ob/obmice with daily doses of TNP-470 ranging from 2.5 mg/kg to 10 mg/kg.Brakenhielm describes prevention of obesity at TNP-470 doses of 15 or 20mg/kg every other day, “The Angiogenesis Inhibitor, TNP-470, PreventsDiet-Induced and Genetic Obesity in Mice” Circulation Research 94:1579-1588, 2004. Kim, et al., in the “Assessment of the anti-obesityeffects of the TNP-470 analog, CKD-732” J Molecular Endocrinology 38,455-465, 2007 describe weight loss in C57BL/6J mice and SD rats at dosesof 5 mg/kg/day. Lijnen et al. “Fumagillin reduces adipose tissueformation in murine models of nutritionally induced obesity” Obesity 12,2241-2246, 2010 describes oral delivery of 1 mg/kg fumagillin dailyresulting in weight loss in C57BL/6 mice.

One of these derivatives, chloroacetylcarbamoylfumagillol (TNP-470) hasbeen extensively studied. (See H Mann-Steinberg, et al., “TNP-470: TheResurrection of the First Synthetic Angiogenesis Inhibitor”, Chapter 35in Folkman and Figg, Angiogenesis: An Integrative Approach from Scienceto Medicine, Springer NY (2008).) TNP-470 has shown activity againstmany cancers including lung cancer, cervical cancer, ovarian cancer,breast cancer and colon cancer. Because of dose-limiting neurotoxicity,TNP-470 has been tested using multiple dosing regimens, but theseattempts to limit its toxicity have been unsuccessful. Thus, TNP-470 hasbeen found to be too toxic for human use. TNP-470 has a short half-lifeand requires extended intravenous administration for therapeutic use. Ametabolite of TNP-470, carbamoylfumagillol has a half-life of 12 minutesin man. (See Herbst et al., “Safety and Pharmacokinetic Effects ofTNP-470, an Angiogenesis Inhibitor, Combined with Paclitaxel in Patientswith Solid Tumors: Evidence for Activity in Non-Small-Cell Lung Cancer”,Journal of Clinical Oncology 20(22) 4440-4447 (2002). In addition,fumagillin and its derivatives are hydrophobic and difficult toformulate.

Despite the known usefulness of fumagillin derivatives, they have notbeen used successfully as treatments because of the failure to overcomethe problems of the low water solubility, short half-life values, andneurotoxic side-effects of these compounds. TNP-470 in combination withpaclitaxel was determined to have an MTD of 60 mg/m2 dosed three timesper week based on the previously observed dose limiting neuropsychiatrictoxicities Herbst et al., “Safety and pharmacokinetic effects ofTNP-470, an angiogenesis inhibitor, combined with paclitaxel in patientswith solid tumors: evidence for activity in non-small-cell lung Cancer”Journal of Clinical Oncology 20, 4440-4447, 2002. Similarly Shin et al.“A Phase 1 pharmacokinetic and pharmacodynamics study of CKD-732, anantiangiogenic agent, in patients with refractory solid cancer”Investigational New Drugs 28, 650-658, 2010 reports that the MTD ofCKD-732 was 15 mg/m2/day dosed on an every fourth day schedule due toconfusion and insomnia. Accordingly, the compounds of the presentinvention are more potent, show reduced toxicity (less neurotoxic),improved water solubility, more stable, and/or have longer half-life(serum half-life) than presently known fumagillin derivatives.

The phrase “reduced toxicity” as used herein has its ordinary meaning asunderstood by persons of skill in the art. Merely by way of example, andby no means as a limitation on the meaning of the term, theadministration of the fumagillin analog conjugate causes less sideeffects in open field tests with mice, as compared to the fumagillinanalog alone.

The phrase “improved water solubility” has its ordinary meaning asunderstood by persons of skill in the art. Merely by way of example, andby no means as a limitation on the meaning of the term, the followingdescription of the term is informative: an increased amount of afumagillin analog will dissolve in water as a result of its covalentincorporation into a conjugate as compared to the amount of theunconjugated fumagillin analog that will dissolve in water alone.

The phrase “longer half-life” has its ordinary meaning as understood bypersons of skill in the art. Merely by way of example, and by no meansas a limitation on the meaning of the term, the following description ofthe term is informative: any appreciable increase in the length of timerequired to deactivate fumagillin conjugate either in vivo or in vitroas compared to the half-life of the fumagillin analog alone either invivo or in vitro.

Without being bound by any theory, non-enzymatic actions of MetAP2 tosuppress activity of extra-cellular signal regulated kinases 1 and 2(ERK1/2) may be important as may be the binding of eukaryotic initiationfactor, eIF, by MetAP2. Cellular responses to MetAP2 inhibitionreflective of potential ERK-related processes may include suppression ofsterol regulatory element binding protein (SREBP) activity, leading toreduced lipid and cholesterol biosynthesis. Interesting, changes in theexpression patterns of hepatic and adipose tissue genes after prolonged(approximately 9 months) fumagillin exposure suggest that MetAP2inhibition also may alter the relative abundance of factors involved ininflammation, consistent with reduced ERK-dependent cellular processes.The putative mechanism of MetAP2 inhibition leading to mobilization ofadipose depot and catabolism of free fatty acids as energy source by thebody is supported by changes in plasma β-hydroxybutyrate, adiponectin,leptin, and FGF21 observed in previous studies. Elevation in the levelsof key catabolic hormones adiponectin and FGF21, coupled with theappearance of ketone bodies (β-hydroxybutyrate), suggest MetAP2inhibition with the conjugated or modified fumagillin, fumagillol, or ananalog, derivative, salt or ester thereof compounds of the presentinvention stimulates energy expenditure, fat utilization and lipidexcretion. The reduction in leptin observed in previous studies and thestudies provided herein is also consistent with a decrease in totaladipose tissue and negative energy balance. It is also possible that theconjugated or modified fumagillin, fumagillol, or an analog, derivative,salt or ester thereof compounds of the present invention form a covalentbond with MetAP2, thereby irreversibly inhibiting and silencing existingenzyme until a newly produced pool of MetAP2 is generated in targettissues (e.g., liver and adipose tissue.

In certain embodiments, the conjugated or modified fumagillin,fumagillol, or an analog, derivative, salt or ester thereof compounds ofthe present invention, for example have the following formula as shownin Table 1:

TABLE 1 Compound No. Chemical Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

*wherein Polymer has the structure of:

and preferably the structure of:

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term “alkyl” refers to a fully saturated branched or unbranchedcarbon chain radical having the number of carbon atoms specified, or upto 30 carbon atoms if no specification is made. For example, a “loweralkyl” refers to an alkyl having from 1 to 10 carbon atoms, such asmethyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, andthose which are positional isomers of these alkyls. Alkyl of 10 to 30carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, certain cycloalkylshave from 3-10 carbon atoms in their ring structure, and may have 5, 6,or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl”, asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, or from one to six carbon atoms in its backbonestructure such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Throughout the application, certain alkylgroups are lower alkyls. In certain embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

“Alkenyl” refers to any branched or unbranched unsaturated carbon chainradical having the number of carbon atoms specified, or up to 26 carbonatoms if no limitation on the number of carbon atoms is specified; andhaving 1 or more double bonds in the radical. Alkenyl of 6 to 26 carbonatoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,where the unsaturated bond(s) can be located anywhere in the radical andcan have either the (Z) or the (E) configuration about the doublebond(s).

The term “alkynyl” refers to hydrocarbyl radicals of the scope ofalkenyl, but having one or more triple bonds in the radical.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₁,where m and R₁ are described below.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN,or the like.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH₂)_(m)—R₁, wherein m and R₁ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates F, Cl, Br or I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formulae:

wherein R₃, R₅ and R₆ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R₁, or R₃ and R₅ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R₁ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In certain embodiments, only one of R₃or R₅ can be a carbonyl, e.g., R₃, R₅ and the nitrogen together do notform an imide. In certain embodiments, R₃ and R₅ (and optionally R₆)each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₁. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₃ and R₅ is an alkyl group. Incertain embodiments, an amino group or an alkylamine is basic, meaningit has a pK_(a)≧7.00. The protonated forms of these functional groupshave pK_(a)s relative to water above 7.00.

The term “carbonyl” (C(O)) is art-recognized and includes such moietiesas can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₇represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₁ or apharmaceutically acceptable salt, R₈ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R₁, where m and R₁ are as defined above. Where Xis an oxygen and R₇ or R₈ is not hydrogen, the formula represents an“ester”. Where X is an oxygen, and R₇ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₇ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R₈ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₇ or R₈ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R₇ is hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R₈ ishydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R₇ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R₇ is hydrogen, theabove formula represents an “aldehyde” group.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that “substitution” or “substitutedwith” includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₃ and R₅ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is as defined above.

The term “sulfamido” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₂ and R₄ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₁₂ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The term “amino acid” is intended to embrace all compounds, whethernatural or synthetic, which include both an amino functionality and anacid functionality, including amino acid analogs and derivatives. Incertain embodiments, the amino acids contemplated in the presentinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids, which contain amino and carboxyl groups. Naturally occurringamino acids are identified throughout by the conventional three-letterand/or one-letter abbreviations, corresponding to the trivial name ofthe amino acid, in accordance with the following list. The abbreviationsare accepted in the peptide art and are recommended by the IUPAC-IUBcommission in biochemical nomenclature.

By the term “amino acid residue” is meant an amino acid. In general theabbreviations used herein for designating the naturally occurring aminoacids are based on recommendations of the IUPAC-IUB Commission onBiochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). Forinstance Met, Ile, Leu, Ala and Gly represent “residues” of methionine,isoleucine, leucine, alanine and glycine, respectively. By the residueis meant a radical derived from the corresponding α-amino acid byeliminating the OH portion of the carboxyl group and the H portion ofthe α-amino group.

The term “amino acid side chain” is that part of an amino acid residueexclusive of the backbone, as defined by K. D. Kopple, “Peptides andAmino Acids”, W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H-(theside chain of glycine). These side chains are pendant from the backboneCα carbon.

The term “peptide,” as used herein, refers to a sequence of amino acidresidues linked together by peptide bonds or by modified peptide bonds.The term “peptide” is intended to encompass peptide analogs, peptidederivatives, peptidomimetics and peptide variants. The term “peptide” isunderstood to include peptides of any length. Peptide sequences set outherein are written according to the generally accepted conventionwhereby the N-terminal amino acid is on the left, and the C-terminalamino acid is on the right (e.g., H₂N-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-CO₂H).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.Any representation of a particular isomer is merely exemplary (e.g., theexemplification of a trans-isomer, also encompasses a cis-isomer).

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomer.

Synthesis of the Compounds of the Invention

The synthetic processes of the invention can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof

Compounds of the present invention can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3^(rd) edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentinvention.

Compounds of the present invention can be conveniently prepared by avariety of methods familiar to those skilled in the art. The compoundsof the present invention may be prepared according to the schemes andexamples provided herein from commercially available starting materialsor starting materials which can be prepared using literature procedures.The compounds of the present invention, and their synthesis, are furtherdescribed in PCT Publication Nos. WO 2011/150088 and WO 2011/150022.Each of these publications is incorporated by reference in theirentireties for all purposes.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising a compound of the present invention, or pharmaceuticallyacceptable salts, solvates, diastereomers, and polymorphs thereof, and apharmaceutically acceptable carrier or excipient.

As used herein, “pharmaceutically acceptable excipient” or“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin.

Pharmaceutically acceptable carriers include solid carriers such aslactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary liquid carriersinclude syrup, peanut oil, olive oil, water and the like. Similarly, thecarrier or diluent may include time-delay material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or with awax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate orthe like. Other fillers, excipients, flavorants, and other additivessuch as are known in the art may also be included in a pharmaceuticalcomposition according to this invention. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, the pharmaceutical compositioncomprises DMSO.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the compound(s).These salts can be prepared in situ during the final isolation andpurification of the compound(s), or by separately reacting a purifiedcompound(s) in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts, and the like. Representative alkali or alkalineearth salts include the lithium, sodium, potassium, calcium, magnesium,and aluminum salts, and the like. Representative organic amines usefulfor the formation of base addition salts include ethylamine,diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine,and the like (See, for example, Berge et al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19)

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals, substantiallynon-pyrogenic, without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

As used herein, the term “metabolite” means a product of metabolism ofthe compound of present invention, or pharmaceutically acceptable salts,solvates, diastereomers, and polymorphs thereof, that exhibits a similaractivity in vivo to the compound of present invention, orpharmaceutically acceptable salts, solvates, diastereomers, andpolymorphs thereof

As used herein, the term “prodrug” means the compound of presentinvention, or pharmaceutically acceptable salts, solvates,diastereomers, and polymorphs thereof covalently linked to one or morepro-moieties, such as an amino acid moiety or other water solubilizingmoiety. The compound of present invention, or pharmaceuticallyacceptable salts, solvates, diastereomers, and polymorphs thereof may bereleased from the pro-moiety via hydrolytic, oxidative, and/or enzymaticrelease mechanisms. In an embodiment, a prodrug composition of thepresent invention exhibits the added benefit of increased aqueoussolubility, improved stability, and improved pharmacokinetic profiles.The pro-moiety may be selected to obtain desired prodrugcharacteristics. For example, the pro-moiety, e.g., an amino acid moietyor other water solubilizing moiety such as phosphate within R4, may beselected based on solubility, stability, bioavailability, and/or in vivodelivery or uptake. Examples of prodrugs include, but are not limitedto, esters (e.g., acetate, dialkylaminoacetates, formates, phosphates,sulfates and benzoate derivatives) and carbamates (e.g.,N,N-dimethylaminocarbonyl) of hydroxy functional groups, esters (e.g.,ethyl esters, morpholinoethanol esters) of carboxyl functional groups,N-acyl derivatives (e.g., N-acetyl)N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p1-92,Elesevier, New York-Oxford (1985).

Methods of Treatment

The present invention provides methods of inducing or causing weightloss in a subject in need thereof comprising administering at least onecompound of the present invention in a therapeutically effective amountto the subject to induce or cause weight loss. In certain embodiments,the subject is overweight or obese. In certain embodiments, inducing orcausing weight loss is increasing weight loss.

The present invention also provides methods for preventing or delayingweight increase in a subject at risk thereof comprising administering atleast one compound of the present invention in a therapeuticallyeffective amount to the subject to prevent or delay the increase inweight. In certain embodiments, the subject is at risk of becomingoverweight or becoming obese.

The present invention provides methods of treating obesity in a subjectin need thereof comprising administering at least one compound of thepresent invention in a therapeutically effective amount to the subjectto treat or ameliorate obesity.

The present invention also provides methods for preventing or delayingthe development of obesity in a subject at risk thereof comprisingadministering at least one compound of the present invention in atherapeutically effective amount to the subject to prevent or delay thedevelopment of obesity.

The present invention provides methods of treating metabolic syndrome orone or more of the components thereof in a subject in need thereofcomprising administering at least one compound of the present inventionin a therapeutically effective amount to the subject to treat orameliorate metabolic syndrome or one or more of the components thereof

The present invention also provides methods for preventing or delayingthe development of metabolic syndrome or one or more of the componentsthereof in a subject at risk thereof comprising administering at leastone compound of the present invention in a therapeutically effectiveamount to the subject to prevent or delay the development of metabolicsyndrome or one or more of the components thereof

The present invention also provides methods of decreasing body weight ina subject in need thereof comprising administering at least one compoundof the present invention in a therapeutically effective amount to thesubject to decrease body weight. In certain embodiments, the subject isoverweight or obese. In certain embodiments the subject is in need ofreducing excess adipose tissue.

Obesity and being overweight refer to an excess of fat in a subject inproportion to lean body mass. Excess fat accumulation is associated withan increase in size (hypertrophy or steatosis) as well as number(hyperplasia) of adipose tissue cells. Obesity may be due to any cause,whether genetic (e.g. Prader-Willi Syndrome) or environmental. Obesityis variously measured in terms of absolute weight, weight:height ratio,degree of excess body fat, distribution of visceral or subcutaneous fat,and societal and esthetic norms. A common measure of body fat is BodyMass Index (BMI). The BMI refers to the ratio of body weight (expressedin kilograms) to the square of height (expressed in meters). Body massindex may be accurately calculated using the formulas: SI units:BMI=weight(kg)/(height²(m²), or US units:BMI=(weight(lb)*703)/(height²(in²).

As described herein, “overweight” refers to a condition whereby anotherwise healthy adult that has a BMI of 25 kg/m² to 29.9 kg/m². Asdescribed herein, “obese” or “obesity” refers to a condition whereby anotherwise healthy adult that has a BMI of 30 kg/m² or greater. Obesityhas several subcategories. An adult that has a BMI of 35 kg/m² orgreater is referred to as “severely obese” or “severe obesity”. An adultthat has a BMI of ≧40-44.9 kg/m² or and adult that has a BMI of 35 kg/m²or greater and at least one obesity-related health condition is referredto as “morbidly obese” or “morbid obesity”. An adult that has a BMI of45 kg/m² or greater is referred to as “super obese” or “super obesity”.For children, the definitions of overweight and obese take into accountage and gender effects on body fat.

Different countries may define obesity and overweight with differentBMI. The term “obesity” is meant to encompass definitions in allcountries. For example, the increased risks associated with obesityoccur at a lower Body Mass Index (BMI) in Asians. In Asian countries,including Japan, “obesity” refers to a condition whereby a subject withat least one obesity-induced or obesity-related co-morbidity, thatrequires weight reduction or that would be improved by weight reduction,has a BMI greater than or equal to 25.0 kg/m² Ethnic South and CentralAmericans tend to be categorized more closely to Asians than Europeansor North Americans.

BMI does not account for the fact that excess adipose tissue can occurselectively in different parts of the body, and development of adiposetissue can be more dangerous to health in some parts of the body ratherthan in other parts of the body. For example, “central obesity”,typically associated with an “apple-shaped” body, results from excessadiposity especially in the abdominal region, including belly fat andvisceral fat, and carries higher risk of co-morbidity than “peripheralobesity”, which is typically associated with a “pear-shaped” bodyresulting from excess adiposity especially on the hips. Measurement ofwaist/hip circumference ratio (WHR) can be used as an indicator ofcentral obesity. A minimum WHR indicative of central obesity has beenvariously set, and a centrally obese adult typically has a WHR of about0.85 or greater if female and about 0.9 or greater if male.

Assessment of disease is performed using standard methods known in thearts, for example, by monitoring appropriate marker(s). For example, thefollowing markers may be monitored for obesity: body weight, BMI, bodycomposition study, body fat distribution, central fat distribution, foodor calorie intake, behavioral measurement of hunger and satiety,metabolic rate, and obesity-related co-morbidities.

Methods of determining whether a subject is overweight or obese thataccount for the ratio of excess adipose tissue to lean body mass mayinvolve obtaining a body composition of the subject. Body compositioncan be obtained by measuring the thickness of subcutaneous fat inmultiple places on the body, such as the abdominal area, the subscapularregion, arms, buttocks and thighs. These measurements are then used toestimate total body fat with a margin of error of approximately fourpercentage points. Another method is bioelectrical impedance analysis(BIA), which uses the resistance of electrical flow through the body toestimate body fat. Another method is using a large tank of water tomeasure body buoyancy. Increased body fat will result in greaterbuoyancy, while greater muscle mass will result in a tendency to sink.Another method is fan-beam dual energy X-ray absorptiometry (DEXA). DEXAallows body composition, particularly total body fat and/or regional fatmass, to be determined non-invasively. MRI may also be used to determinecomposition non-invasively.

With respect to all methods described herein, reference to the compoundsof the instant invention also include compositions, such aspharmaceutical compositions as described herein, comprising one or moreof these compounds. These compositions may further comprise suitableexcipients, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art. The present invention can beused alone or in combination with other conventional methods oftreatment.

A subject in need of treatment as provided by the present invention mayalso have (i.e., be diagnosed with or suffering from) at least oneobesity-induced or obesity-related co-morbidity, i.e., diseases andother adverse health conditions associated with, exacerbated by, orprecipitated by being overweight or obese. In other embodiments, thesubject may have at least two obesity-induced or obesity-relatedco-morbidities.

Obesity-induced or obesity-related co-morbidities include, but are notlimited to, diabetes, non-insulin dependent diabetes mellitus-type II,impaired glucose tolerance, impaired fasting glucose, dysglycaemia,elevated plasma insulin concentrations, insulin resistance syndrome,hyperlipidemia, dyslipidemia, elevated free fatty acids, hypertension,hyperuricacidemia, gout, coronary artery disease, cardiac disease,myocardial infarction, angina pectoris, microvascular disease, sleepapnea, obstructive sleep apnea, Pickwickian syndrome, fatty liver;cerebral infarction, stroke, cerebral thrombosis, respiratorycomplications, cholelithiasis, gallbladder disease, kidney disease,gastro-esophageal reflux, stress urinary incontinence, arteriosclerosis,heart disease, abnormal heart rhythms, heart arrhythmias, transientischemic attack, orthopedic disorders, osteoarthritis, arthritisdeformans, lumbodynia, emmeniopathy, hormonal imbalances,endocrinopathies and infertility. In particular, co-morbidities include:hypertension, hyperlipidemia, dyslipidemia, glucose intolerance,cardiovascular disease, sleep apnea, diabetes mellitus, and otherobesity-related conditions.

The present invention provides, in addition to treating obesity orinducing, causing or increasing weight loss (decreasing weight) in asubject in need thereof, methods of treating one or more of theseobesity-induced or obesity-related co-morbidities in a subject sufferingfrom said co-morbidities comprising administering at least one compoundof the present invention in a therapeutically effective amount to thesubject to treat or ameliorate obesity or decrease body weight, andtreat or ameliorate one or more of obesity-induced or obesity-relatedco-morbidities.

The present invention provides methods for treating metabolic disordersor metabolic syndrome in a subject in need thereof wherein said syndromeis characterized by a group of metabolic risk factors including: 1)abdominal obesity (excessive fat tissue in and around the abdomen); 2)atherogenic dyslipidemia (high triglycerides; low HDL cholesterol andhigh LDL cholesterol or a low HDL:LDL ratio); 3) elevated bloodpressure; 4) insulin resistance or glucose intolerance; 5) aprothrombotic state (e.g., high fibrinogen or plasminogen activatorinhibitor-1 in the blood); 6) a proinflammatory state (e.g., elevatedCRP in the blood); and 7) pre-diabetes or type 2 diabetes. The presentinvention can treat metabolic disease(s) alone or in combination withtreating obesity or inducing, causing or increasing weight loss.

The present invention also provides, in addition to treating obesity orinducing, causing or increasing weight loss in a subject in needthereof, methods of treating, decreasing or improving one or morecardiometabolic risk factors selected from but not limited to the groupconsisting of plasma triglyceride levels, LDL-cholesterol levels,C-reactive protein (CRP) levels, and blood pressure (systolic and/ordiastolic) in a subject suffering from said risk factors comprisingadministering at least one compound of the present invention in atherapeutically effective amount to the subject to treat or ameliorateobesity or decrease body weight, and treat or ameliorate one or more ofrisk factors.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, may beadministered in combination with a second active agent. The secondactive agent may also be conjugated to a polymer.

Contemplated second active agents include those administered to treattype 2 diabetes such as sulfonylureas (e.g., chlorpropamide, glipizide,glyburide, glimepiride); meglitinides (e.g., repaglinide andnateglinide); biguanides (e.g., metformin); thiazolidinediones(rosiglitazone, troglitazone, and pioglitazone); glucagon-like 1 peptidemimetics (e.g. exenatide and liraglutide); sodium-glucose cotransporterinhibitors (e.g., dapagliflozin), dipeptidyl peptidase 4 inhibitors(e.g. gliptins), sodium-glucose linked transporter inhibitors, renininhibitors, and alpha-glucosidase inhibitors (e.g., acarbose andmeglitol), and/or those administered to treat cardiac disorders andconditions, such as hypertension, dyslipidemia, ischemic heart disease,cardiomyopathy, cardiac infarction, stroke, venous thromboembolicdisease and pulmonary hypertension, which have been linked to overweightor obesity, for example, chlorthalidone; hydrochlorothiazide;indapamide, metolazone; loop diuretics (e.g., bumetanide, ethacrynicacid, furosemide, lasix, torsemide); potassium-sparing agents (e.g.,amiloride hydrochloride, spironolactone, and triamterene); peripheralagents (e.g., reserpine); central alpha-agonists (e.g., clonidinehydrochloride, guanabenz acetate, guanfacine hydrochloride, andmethyldopa); alpha-blockers (e.g., doxazosin mesylate, prazosinhydrochloride, and terazosin hydrochloride); beta-blockers (e.g.,acebutolol, atenolol, betaxolol, nisoprolol fumarate, carteololhydrochloride, metoprolol tartrate, metoprolol succinate, Nadolol,penbutolol sulfate, pindolol, propranolol hydrochloride, and timololmaleate); combined alpha- and beta-blockers (e.g., carvedilol andlabetalol hydrochloride); direct vasodilators (e.g., hydralazinehydrochloride and minoxidil); calcium antagonists (e.g., diltiazemhydrochloride and verapamil hydrochloride); dihydropyridines (e.g.,amlodipine besylate, felodipine, isradipine, nicardipine, nifedipine,and nisoldipine); ACE inhibitors (benazepril hydrochloride, captopril,enalapril maleate, fosinopril sodium, lisinopril, moexipril, quinaprilhydrochloride, ramipril, trandolapril); angiotensin II receptor blockers(e.g., losartan potassium, valsartan, and Irbesartan); and combinationsthereof, as well as statins such as mevastatin, lovastatin, pravastatin,simvastatin, velostatin, dihydrocompactin, fluvastatin, atorvastatin,dalvastatin, carvastatin, crilvastatin, bevastatin, cefvastatin,rosuvastatin, pitavastatin, and glenvastatin., typically for treatmentof dyslipidemia.

Other second active agents that may be co-administered (e.g.sequentially or simultaneously) include agents administered to treatischemic heart disease including statins, nitrates (e.g., IsosorbideDinitrate and Isosorbide Mononitrate), beta-blockers, and calciumchannel antagonists, agents administered to treat cardiomyopathyincluding inotropic agents (e.g., Digoxin), diuretics (e.g.,Furosemide), ACE inhibitors, calcium antagonists, anti-arrhythmic agents(e.g., Sotolol, Amiodarone and Disopyramide), and beta-blockers, agentsadministered to treat cardiac infarction including ACE inhibitors,Angiotensin II receptor blockers, direct vasodilators, beta blockers,anti-arrhythmic agents and thrombolytic agents (e.g., Alteplase,Retaplase, Tenecteplase, Anistreplase, and Urokinase), agentsadministered to treat strokes including anti-platelet agents (e.g.,Aspirin, Clopidogrel, Dipyridamole, and Ticlopidine), anticoagulantagents (e.g., Heparin), and thrombolytic agents, agents administered totreat venous thromboembolic disease including anti-platelet agents,anticoagulant agents, and thrombolytic agents, agents administered totreat pulmonary hypertension include inotropic agents, anticoagulantagents, diuretics, potassium (e.g., K-dur), vasodilators (e.g.,Nifedipine and Diltiazem), Bosentan, Epoprostenol, and Sildenafil,agents administered to treat asthma include bronchodilators,anti-inflammatory agents, leukotriene blockers, and anti-Ige agents.Particular asthma agents include Zafirlukast, Flunisolide,Triamcinolone, Beclomethasone, Terbutaline, Fluticasone, Formoterol,Beclomethasone, Salmeterol, Theophylline, and Xopenex, agentsadministered to treat sleep apnea include Modafinil and amphetamines,agents administered to treat nonalcoholic fatty liver disease includeantioxidants (e.g., Vitamins E and C), insulin sensitizers (Metformin,Pioglitazone, Rosiglitazone, and Betaine), hepatoprotectants, andlipid-lowering agents, agentsadministered to treat osteoarthritis ofweight-bearing joints include Acetaminophen, non-steroidalanti-inflammatory agents (e.g., Ibuprofen, Etodolac, Oxaprozin,Naproxen, Diclofenac, and Nabumetone), COX-2 inhibitors (e.g.,Celecoxib), steroids, supplements (e.g. glucosamine and chondroitinsulfate), and artificial joint fluid, agents administered to treatPrader-Willi Syndrome include human growth hormone (HGH), somatropin,and weight loss agents (e.g., Orlistat, Sibutramine, Methamphetamine,Ionamin, Phentermine, Bupropion, Diethylpropion, Phendimetrazine,Benzphetermine, and Topamax), agents administered to treat polycysticovary syndrome include insulin-sensitizers, combinations of syntheticestrogen and progesterone, Spironolactone, Eflornithine, and Clomiphene,agents administered to treat erectile dysfunction includephosphodiesterase inhibitors (e.g., Tadalafil, Sildenafil citrate, andVardenafil), prostaglandin E analogs (e.g., Alprostadil), alkaloids(e.g., Yohimbine), and testosterone, agents administered to treatinfertility include Clomiphene, Clomiphene citrate, Bromocriptine,Gonadotropin-releasing Hormone (GnRH), GnRH agonist, GnRH antagonist,Tamoxifen/nolvadex, gonadotropins, Human Chorionic Gonadotropin (HCG),Human Menopausal Gonadotropin (HmG), progesterone, recombinant folliclestimulating hormone (FSH), Urofollitropin, Heparin, Follitropin alfa,and Follitropin beta, agents administered to treat obstetriccomplications include Bupivacaine hydrochloride, Dinoprostone PGE2,Meperidine HCl, Ferro-folic-500/iberet-folic-500, Meperidine,Methylergonovine maleate, Ropivacaine HCl, Nalbuphine HCl, OxymorphoneHCl, Oxytocin, Dinoprostone, Ritodrine, Scopolamine hydrobromide,Sufentanil citrate, and Oxytocic, agents administered to treatdepression include serotonin reuptake inhibitors (e.g., Fluoxetine,Escitalopram, Citalopram, Paroxetine, Sertraline, and Venlafaxine);tricyclic antidepressants (e.g., Amitriptyline, Amoxapine, Clomipramine,Desipramine, Dosulepin hydrochloride, Doxepin, Imipramine, Iprindole,Lofepramine, Nortriptyline, Opipramol, Protriptyline, and Trimipramine);monoamine oxidase inhibitors (e.g., Isocarboxazid, Moclobemide,Phenelzine, Tranylcypromine, Selegiline, Rasagiline, Nialamide,Iproniazid, Iproclozide, Toloxatone, Linezolid, Dienolide kavapyronedesmethoxyyangonin, and Dextroamphetamine); psychostimulants (e.g.,Amphetamine, Methamphetamine, Methylphenidate, and Arecoline);antipsychotics (e.g., Butyrophenones, Phenothiazines, Thioxanthenes,Clozapine, Olanzapine, Risperidone, Quetiapine, Ziprasidone,Amisulpride, Paliperidone, Symbyax, Tetrabenazine, and Cannabidiol); andmood stabilizers (e.g., Lithium carbonate, Valproic acid, Divalproexsodium, Sodium valproate, Lamotrigine, Carbamazepine, Gabapentin,Oxcarbazepine, and Topiramate), agents administered to treat anxietyinclude serotonin reuptake inhibitors, mood stabilizers, benzodiazepines(e.g., Alprazolam, Clonazepam, Diazepam, and Lorazepam), tricyclicantidepressants, monoamine oxidase inhibitors, and beta-blockers, andother weight loss agents, including serotonin and noradrenergicre-uptake inhibitors; noradrenergic re-uptake inhibitors; selectiveserotonin re-uptake inhibitors; and intestinal lipase inhibitors.Particular weight loss agents include orlistat, sibutramine,methamphetamine, ionamin, phentermine, bupropion, diethylpropion,phendimetrazine, benzphetermine, and topamax.

The present invention also provides methods of decreasing adipocytes ina subject in need thereof comprising administering at least one compoundof the present invention in a therapeutically effective amount to thesubject to decrease adipocytes or adipose tissue. The present inventionalso provides methods of preventing an increase in adipocytes in asubject at risk thereof comprising administering at least one compoundof the present invention in a therapeutically effective amount to thesubject to prevent an increase in adipocytes. Decreasing adipocytesmeans decreasing the number or decreasing the size (fat content) of theadipocytes. Preventing an increase in adipocytes means decreasing ormaintaining the number or decreasing or maintaining the size of theadipocytes. In certain embodiments, administration of the compounds ofthe present invention shrink the adipocytes in the subject in needthereof. The adipose tissue can be white adipose tissue or brown adiposetissue.

The present invention also provides methods of decreasing food intake ina subject in need thereof comprising administering at least one compoundof the present invention in a therapeutically effective amount to thesubject to decrease food intake.

A reduction in food intake means a decrease in daily food intake. Adecrease in daily food intake can be about a 5% decrease to about a 50%decrease (e.g., about 5%, about 10%, about 20%, about 30%, about 40% orabout 50%). Based on a 2000 kcal daily diet, the decrease is about 100kcal to about 1000 kcal decrease per day (e.g., about 100 kcal, about200 kcal, about 400 kcal, about 600 kcal, about 800 kcal or about 1000kcal).

The present invention also provides methods for reducing a sense ofhunger in a subject in need thereof comprising administering at leastone compound of the present invention in a therapeutically effectiveamount to the subject to reduce a sense of hunger. The subject may alsohave a decrease in food intake.

Sense of hunger can be assessed in a fasted state using a 10-pointvisual analog scale (VAS), which is well utilized in appetite research.See, Flint et al. Int. J. Obes. Relat. Metab. Disord. 24(1): 38-48,2000. Specifically, subjects are asked to rate their overall sense ofhunger for the previous 2 days on a scale of 1-10, where 10 wasextremely hungry and 1 was not hungry at all.

The methods of present invention can also decrease waist circumferencein a subject in need thereof. Waist circumference is assessed by using atape measure placed around the abdomen 1 cm above the iliac crest. Thesubjects of the present invention may have a decrease in waistcircumference from about 1 inch to about 20 inches (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 inches).

In the methods of the present invention, administration of the compoundsresults in decreased body fat and a substantial maintenance of musclemass in said patient. In certain embodiments, upon administration, fatoxidation is enhanced in a patient as compared to a patient on arestricted food intake diet alone. For example, provided herein is amethod of decreasing body fat in a patient in need thereof. Such apatient may retain substantially more muscle mass as compared to bodyfat reduction in a patient using an energy restricted diet alone.

The present invention also provides methods for improving surgicaloutcome in a subject in need thereof comprising administering, prior tosurgery, at least one compound of the present invention in atherapeutically effective amount to the subject to improve surgicaloutcome. In certain embodiments, administration reduces liver and/orabdominal fat in said patient and improves surgical outcome. In certainembodiments, the surgery is non-acute surgery. Such surgeries mayinclude bariatric surgery, cardiovascular surgery, abdominal surgery, ororthopedic surgery.

A “patient” or “subject” as recited herein can mean either a human ornon-human subject. In certain embodiments, the subject is a vertebrate.In certain embodiments, the vertebrate is a mammal. Mammals alsoinclude, but are not limited to, farm animals, sport animals, pets,primates (including humans), horses, dogs, cats, mice and rats. Incertain embodiments, the mammal is a human.

As used herein, a “subject in need thereof” is a subject that isoverweight or obese (who may or may not have one or moreco-morbidities), or a subject having an increased risk of becomingoverweight or developing obesity relative to the population at large. Incertain aspects, a subject in need thereof is obese having a BMI of 30kg/m² or greater. In certain aspects, the subject in need thereof is asubject that is overweight or obese or having an increased risk ofbecoming overweight or developing obesity relative to the population atlarge who is not suffering from, or is not diagnosed with, a disorderselected from the group consisting of cancer, hyper-proliferativedisorder, retinal neovascularization due to macular degeneration,psoriasis and pyogenic granuloma, rheumatoid, immune and degenerativearthritis.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improving, lessening severity, alleviation of one or moresymptoms associated with a disease. For obesity, beneficial or desiredclinical results include any one or more of the following: reducing ormaintaining body weight; controlling (including reducing) food intake orcalorie intake; increasing metabolic rate or inhibiting reduction ofmetabolic rate; and improving, lessening severity, and/or alleviatingany of the disorders associated with obesity, such as diabetes,non-insulin dependent diabetes mellitus, hyperglycemia, low glucosetolerance, insulin resistance, lipid disorder, dyslipidemia,hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, abdominalobesity, eating disorder, metabolic syndrome, hypertension,osteoarthritis, myocardial infarction, fatty liver disease,steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholicfatty liver disease (NAFLD), stroke and other associated diseases;increasing the quality of life of those suffering from the obesity,and/or prolonging lifespan.

As used herein, “delaying” development of obesity means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, one outcome of delaying development may bereducing the body weight of a subject at risk of obesity relative tothat subject's body weight immediately before the administration of thecompositions described herein. Another outcome of delaying developmentmay be preventing regain of body weight previously lost as a result ofdiet, exercise, or pharmacotherapy. Another outcome of delayingdevelopment may be preventing obesity from occurring if the treatment isadministered prior to the onset of obesity in a subject at risk ofobesity. Another outcome of delaying development may be decreasing theoccurrence and/or severity of obesity-related disorders if the treatmentis administered prior to the onset of obesity in a subject at risk ofobesity.

An individual “at risk” of obesity may or may not have detectabledisease, and may or may not have displayed detectable disease prior tothe treatment methods described herein. “At risk” denotes that anindividual has one or more so-called risk factors, which are measurableparameters that correlate with development of obesity. An individualhaving one or more of these risk factors has a higher probability ofbeing obese than an individual without these risk factor(s). These riskfactors include, but are not limited to, age, diet, physical inactivity,metabolic syndrome, family history of obesity, ethnicity, hereditarysyndromes, history of previous disease (e.g. eating disorder, metabolicsyndrome, and obesity), presence of precursor disease (e.g.,overweight). For example, an otherwise healthy individual with a BMI of25.0 to less than 30.0 kg/m² or an individual with at least oneco-morbidity with a BMI of 25.0 kg/m² to less than 27.0 kg/m² is at riskof obesity.

“Development” of obesity means the onset and/or progression of thedisease within an individual (which can be different embodiments of theinvention). Obesity development can be detectable using standardclinical techniques as described herein. However, development alsorefers to disease progression that may be initially undetectable. Forpurposes of this invention, progression refers to the biological courseof the disease state, in this case, as determined by assessing heightand weight for estimating BMI, measuring waist circumference, assessingco-morbidities, as well as the onset and/or worsening of obesitycomplications such as arteriosclerosis, Type II diabetes, polycysticovary disease, cardiovascular diseases, osteoarthritis, dermatologicaldisorders, hypertension, insulin resistance, hypercholesterolemia,hypertriglyceridemia, and cholelithiasis. A variety of these diagnostictests are known in the art. “Development” includes occurrence,recurrence, and onset. As used herein “onset” or “occurrence” of obesityincludes initial onset and and/or recurrence.

As used herein, “controlling body weight” or “improvement in bodyweight” refers to reducing or maintaining the body weight in anindividual (as compared to the level before treatment). In someembodiments, the body weight is generally maintained within the normalrange. The body weight may be reduced by reducing the calorie intakeand/or reducing the body fat accumulation. In some embodiments, the bodyweight is reduced at least about any of 3%, 4%, 5%, 10%, 20%, 30%, 40%,or 50% in the individual as compared to the level before treatment.

As used herein, “controlling food intake” refers to reducing ormaintaining the food intake in an individual (as compared to the levelbefore treatment). In some embodiments, the food intake is generallymaintained in the normal range. In some embodiments, the food intake isreduced by about any of 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50%, in theindividual as compared to the level before treatment.

A “therapeutically effective amount” of a compound, with respect to usein treatment, refers to an amount of a compound in a preparation which,when administered as part of a desired dosage regimen (to a mammal,preferably a human) alleviates a symptom, ameliorates a condition, orslows or prevents the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment. A “therapeutically effectiveamount” is synonymous with “efficacious dose”.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect beneficial or desired results. For prophylactic use, beneficialor desired results include results such as eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingintensity, duration, or frequency of attack of the disease, anddecreasing one or more symptoms resulting from the disease (biochemical,histological and/or behavioral), including its complications andintermediate pathological phenotypes presenting during development ofthe disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication, and/or delaying theprogression of the disease of patients. An effective dosage can beadministered in one or more administrations. For purposes of thisinvention, an effective dosage of drug, compound, or pharmaceuticalcomposition is an amount sufficient to accomplish prophylactic ortherapeutic treatment either directly or indirectly. As is understood inthe clinical context, an effective dosage of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved. For example, aneffective amount of a compound of the present invention for treatingobesity is an amount sufficient to treat or ameliorate one or moresymptoms associated with obesity. An “effective amount” is an amountsufficient to result in one or more of the following (which can alsocorrespond to various embodiments of the invention): decreasing,reducing or controlling body weight, decreasing, reducing or controllingfood intake, increasing metabolic rate, decreasing one or more symptomsresulting from the diseases associated with obesity, increasing thequality of life of those suffering from the obesity, and/or prolonginglifespan.

In providing a subject with one or more of the compounds describedherein, the dosage of administered compound(s) will vary depending uponsuch factors as the subject's age, weight, height, sex, general medicalcondition, previous medical history, disease progression, route ofadministration, formulation and the like.

Dosages for a compound of the present invention may be determinedempirically in individuals who have been given one or moreadministration(s). Individuals are given incremental dosages of acompound of the present invention. To assess efficacy of a compound ofthe present invention, markers of the disease state can be monitored. Itwill be apparent to one of skill in the art that the dosage will varydepending on the individual, the stage of the disease (e.g., stage ofobesity), and the past and concurrent treatments being used.

Toxicity and therapeutic efficacy of compounds of the present inventioncan be determined by standard pharmaceutical procedures in experimentalanimals. Toxic doses may be determined as the maximum tolerated dose(MTD) or alternatively the LD50 (the dose lethal to 50% of thepopulation). Efficacious doses may be determined as the ED50 (the dosetherapeutically effective in 50% of the population) or dose required toprovide some average amount of change in an animal (e.g. the doserequired to provide an average reduction in systolic blood pressure of10 mm Hg in a group of subjects).

Ideally, the efficacious and toxic doses may be determined in the samespecies. However if they are determined in different species, allometricscaling may be used to translate the efficacious or toxic dose toanother species. The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50. Incomparing mice to rats, the commonly accepted scaling factor is 2; therat dose is estimated to be one-half the dose in mice. Thus if the toxicdose in a rat is 100 mg/kg and the efficacious dose in a mouse is 1mg/kg, the therapeutic index in the rat may be calculated as efficaciousdose in rat equals 1 mg/kg/2 or 0.5 mg/kg and the therapeutic index is200. FDA defines a drug as having a narrow therapeutic range if: (a)less than 2-fold difference between median lethal and median effectivedose, or (b) less than 2-fold difference between minimum toxic andminimum effective concentrations in the blood.

Compounds of the present invention which exhibit large therapeuticindices are preferred. While compounds of the present invention thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such compounds of the present invention tothe site of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from animal studies can be used in formulating a rangeof dosage for use in humans. The dosage of such compounds of the presentinvention lies preferably within a range of circulating concentrationsthat include the efficacious dose range with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For compounds of thepresent invention with a MW less than 1000, the therapeuticallyeffective dose can be estimated initially from cell culture assays whileanimal models will provide a better estimation of dose for conjugateswhere the linker requires cleavage to release an active moiety. Suchinformation can be used to more accurately determine useful doses inhumans. It is well known in the art that polymer conjugation dilutes theactivity of the active moiety (polymer is a diluent). This isexemplified in the mouse dosing model of the anti-cancer drugs shown inthe following Table.

Conjugate Dose Parent Drug Drug Dose (mg/kg) Conjugate (mg/kg) TNP-47030 (qod) XMT-1107 800 Docetaxel 12 (Q4d) Opaxio 480 CPT-11 20 (q2d)EZN-2208 145 (q2d) Doxorubicin 5 (q4d) PK1 62 (q7d) Carboplatin 60 (Qd)AP-5356 2200 

Thus, it is well understood that polymer conjugation increases clinicaldoses, where therapeutic index is not improved. This is exemplified inthe human dosing model of the anti-cancer drugs shown in the followingTable.

Conjugate Dose Parent Drug Drug Dose (mg/m²) Conjugate (mg/m²) TNP-470180 XMT-1107 tbd Paclitaxel 175 Opaxio 473 CPT-11 125 EZN-2208 260Doxorubicin 75 PK1 280 Oxaliplatin 85 AP-5346 6400

The polymer conjugate and modified compounds of the present inventionsurprisingly provide superior efficacy and lower toxicity when comparedto the unconjugated and or unmodified parent drug/active moiety.

For example, the fumagillol conjugates and modified fumagillol compoundsof the present invention are surprisingly superior to fumagillol smallmolecules as they provide increased weight reduction in DIO mice atequivalent molar doses. The compounds of the present invention may beused at lower molar doses and with less frequent dosing to provideequivalent weight loss. Lower molar doses and reduced dosing frequencyreduce systemic drug exposure and systemic drug toxicity. Additionally,the fumagillol conjugates and modified fumagillol compounds of thepresent invention provide the following action similar to fumagillolsmall molecules: preferential loss of fat in DIO mice and reduction infood consumption.

Traditional polymer conjugates dilute activity, increase doses by 5-20×and provide little change in therapeutic index (<2×). In contrast, thepolymer conjugate compounds of the present invention surprisingly andunexpectedly provide an enhanced therapeutic index (order of magnitudeimprovement) and demonstrate increased activity at a reduced dose.

In the methods of the present invention, the polymer conjugate compoundsof the present invention surprisingly demonstrate less frequent doseadministration (e.g., q4d, dosing every fourth day, q7d, dosing everyseventh day, q8d, dosing every eighth day), doses which are decreased atleast 84 mole % fumagillol equivalent, reduced AUC in non-targetcompartments while therapeutic index is increased (>10×).

In another embodiment, provided herein are effective dosages, e.g. adaily dosage of a compound of the present invention. For example,provided here are methods that include administering doses of a compoundof the present invention that are effective for weight loss. Forexample, contemplated dosage of a compound of the present invention inthe methods described herein may include administering a doseindependent of body weight of about 200 mg/day, about 80 mg/day, about40 mg/day, about 20 mg/day, about 10 mg/day, about 5 mg/day, about 3mg/day, about 2 mg/day, about 1 mg/day, about 0.5 mg/day, about 0.2mg/day, about 0.05 mg/day, about 0.01 mg/day, or about 0.001 mg/day.

An effective amount of the drug for weight loss in a patient may also bedosed based on body weight or surface area and be about 0.0001 mg/kg toabout 5 mg/kg of body weight per day. For example, a contemplated dosagemay be from about 0.001 to 5 mg/kg of body weight per day, about 0.001mg/kg to 1 mg/kg of body weight per day, about 0.001 mg/kg to 0.1 mg/kgof body weight per day, about 0.001 to about 0.010 mg/kg of body weighta day or about 0.007 mg/kg of body weight a day.

The compounds of the present invention can be administered in an amountsufficient to reduce the body weight of the patient by about 0.5 kg/weekto about 1 kg/week (or about 0.5% of body weight per week to about 1% ofbody weight per week). In certain embodiments, the weekly reduction inbody weight occurs for the duration of the treatment.

Administration of a compound of the present invention in accordance withthe method in the present invention can be continuous or intermittent,depending, for example, upon the recipient's physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a compound of the present invention may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs or until sufficient therapeutic levels areachieved. For example, dosing from one to five times a week iscontemplated. In certain embodiments, a compound of the presentinvention is administered about every fourth day. Other dosing regimensinclude a regimen of, 1 to 5 times per week, every three to four days,or less frequently. In some embodiments, a compound of the presentinvention is administered about once per week, once every two weeks, orabout 1 to 4 times per month depending on the duration of the responseto drug administration. Intermittent dosing regimen with staggereddosages spaced by 2 days up to 7 days or even 14 days may be used. Insome embodiments, treatment may start with a daily dosing and laterchange to weekly even monthly dosing. The progress of this therapy iseasily monitored by conventional techniques and assays, or by measuringMetAP2 as described in U.S. Pat. No. 6,548,477.

Frequency of administration may be determined and adjusted over thecourse of therapy. For example, frequency of administration may bedetermined or adjusted based on the type and severity of the disease tobe treated, whether the agent is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the agent, and the discretion of the attendingphysician. Typically the clinician will administer a compound of thepresent invention until a dosage is reached that achieves the desiredresult

Treatment can be continued for as long or as short a period as desired.A suitable treatment period can be, for example, at least about oneweek, at least about four weeks, at least about one month, at leastabout six months, at least about 1 year, at least about 2 years, orindefinitely. A treatment period can terminate when a desired result,for example a weight loss target, is achieved. For example, when loss ofabout 5% body weight, about 10% body weight, about 20% body weight,about 30% body weight or more has been achieved. A treatment regimen caninclude a corrective phase, during which a compound of the presentinvention is administered in dose, or dosing frequency, sufficient toprovide reduction of excess adiposity is administered, followed by amaintenance phase, during which a lower compound dose, or decreaseddosing frequency, sufficient to prevent re-development of excessadiposity is administered.

The compounds, or pharmaceutically acceptable salts, esters or pro-drugsthereof (or pharmaceutical compositions thereof) can be administered byany means known in the art. For example, the compounds or compositionsof the present invention are administered orally, nasally,transdermally, topically, pulmonary, inhalationally, buccally,sublingually, intraperintoneally, subcutaneously, intramuscularly,intravenously, rectally, intrapleurally, intrathecally and parenterally.Administration can be systemic, e.g., intravenous administration, orlocalized. In certain embodiments, the route of administration may beintravenous, intramuscular, subcutaneous, intradermal, intraperitoneal,intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, andthe like. In certain embodiments, the compound is administeredsubcutaneously.

In one aspect, the compounds of the present invention, orpharmaceutically acceptable salts, solvates, diastereomers, andpolymorphs thereof, are administered in a suitable dosage form orformulation prepared by combining a therapeutically effective amount(e.g., an efficacious level sufficient to achieve the desiredtherapeutic effect) of the compound of the present invention, orpharmaceutically acceptable salts, solvates, diastereomers, andpolymorphs thereof (as an active ingredient) with standardpharmaceutical carriers or diluents according to conventional procedures(i.e., by producing a pharmaceutical composition of the invention).These procedures may involve mixing, granulating, and compressing ordissolving the ingredients as appropriate to attain the desiredpreparation.

Parenteral dosage forms may be prepared by any means known in the art.For example, sterile injectable aqueous or oleaginous suspensions may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents.

Oral dosage forms, such as capsules, tablets, pills, powders, andgranules, may be prepared using any suitable process known to the art.For example, the compounds of the present invention may be mixed withenteric materials and compressed into tablets. Alternatively,formulations of the invention are incorporated into chewable tablets,crushable tablets, tablets that dissolve rapidly within the mouth, ormouth wash.

For pulmonary (e.g., intrabronchial) administration, the compounds ofthe present invention can be formulated with conventional excipients toprepare an inhalable composition in the form of a fine powder oratomizable liquid. For ocular administration, the compounds of thepresent invention can be formulated with conventional excipients, forexample, in the form of eye drops or an ocular implant. Among excipientsuseful in eye drops are viscosifying or gelling agents, to minimize lossby lacrimation through improved retention in the eye.

Liquid dosage forms for oral or other administration include, but arenot limited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeagent(s), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the ocular, oral, or other systemically-delivered compositionscan also include adjuvants such as wetting agents, and emulsifying andsuspending agents.

Commercially available nebulizers for liquid formulations, including jetnebulizers and ultrasonic nebulizers are useful for administration.Liquid formulations can be directly nebulized and lyophilized powder canbe nebulized after reconstitution. Alternatively, the compounds of thepresent invention can be aerosolized using a fluorocarbon formulationand a metered dose inhaler, or inhaled as a lyophilized and milledpowder.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition may include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants, or patches. Theactive agent is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. For example, cutaneous routes of administration are achievedwith aqueous drops, a mist, an emulsion, or a cream.

Transdermal patches may have the added advantage of providing controlleddelivery of the active ingredients to the body. Such dosage forms can bemade by dissolving or dispensing the compound in the proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the compound in a polymermatrix or gel.

Compositions for rectal or vaginal administration may be suppositorieswhich can be prepared by mixing the compounds of the present inventionwith suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active agent(s).Alternatively, contemplated formulations can be administered by releasefrom a lumen of an endoscope after the endoscope has been inserted intoa rectum of a subject.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3d ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18th edition (1990). Thesetexts can, of course, also be referred to in making or using an aspectof the invention.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

General Procedures

Tangential Flow Filtration (TFF) was used to purify the polymer productsof the invention. TFF was performed with a Pall Minimate™ Capsule andMinimate™ TFF system according to the manufacturer's instructions.Either a Minimate TFF Capsule with 5 kDa Omega membrane (5K) or MinimateTFF Capsule with 10 kDa Omega membrane (10K) cartridge was used forpurification. In all cases, the permeate was discarded and the retentatelyophilized to yield the polymer product. Structures of products wereconfirmed by ¹H NMR, small molecules were also characterized by MS.Polymer weights reported in the examples were not corrected for watercontent.

Carbamoylfumagillol and chloroacetylcarbamoylfumagillol can be preparedaccording to the methods disclosed in U.S. Pat. No. 5,166,172(Kishimoto, et al., incorporated herein by reference). p-Nitrophenylfumagill-6-yl carbonate can be prepared according to publishedprocedures. (See Han, C. et al. Biorg. Med. Chem. Lett. 2000, 10,39-43). MA-GFLG-ONp can be prepared according to the methods disclosedin U.S. Pat. No. 5,258,453 (Kopecek et al. incorporated herein byreference.)

Synthesis of poly(HPMA-co-MA-GFLG-ONp)

A mixture of hydroxypropylmethacrylamide (HPMA, 22.16 g, 155 mmol),N-methyacryl-gly-phe-leu-gly p-nitrophenyl ester (MA-GFLG-ONp, 10.00 g,17.19 mmol), AIBN (1.484 g, 9.037 mmol) and acetone (225 g) was degassed(freeze, pump, thaw, 4 cycles). The resulting reaction mixture wasstirred at 50° C. for 48 hours, then cooled to room temperature. Thedesired product was purified by trituration with acetone, then driedunder vacuum to yield 17.6 g of poly(HPMA-co-MA-GFLG-ONp) as a whitesolid. The structure was verified by ¹H NMR and the product shown to befree from substantial impurities (e.g., p-nitrophenol). Based on UVabsorbance, the copolymer contained 0.47 mmoles of p-nitrophenyl esterper gram of polymer. The copolymer of this example is used in most ofthe subsequent examples. A wide range of copolymers based on differentmonomers and/or monomer ratios may be made following this procedure byadjusting the stoichiometry and/or using different monomers.

Synthesis of poly(HPMA-co-MA-GFLG-OH)

Poly(HPMA-co-MA-GFLG-ONp) (700 mg) was added portionwise to a solutionof 0.1 M NaOH (11.3 mL) at 0° C. The yellow reaction mixture was stirredat 0° C. for 0.5 hours, then at room temperature for 4 hours. One-halfof the solution was acidified with 0.1 M HCl to pH=6. The aqueous phasewas extracted with ethyl acetate to remove excess p-nitrophenol. Theaqueous phase was lyophilized to afford poly(HPMA-co-MA-GFLG-OH) as acolorless solid (360 mg).

Synthesis of poly(HPMA-co-MA-GG-ONp)

A mixture of hydroxypropylmethacrylamide (HPMA, 82.5 g),N-methyacryl-gly-gly p-nitrophenyl ester (MA-GG-ONp, 16.8 g), AIBN (5.7g,) and acetone (875 g) was sparged with argon for 90 min. The resultingreaction mixture was stirred at 50° C. for 48 hours, then cooled to roomtemperature. The desired product was purified by trituration withacetone, then dried under vacuum to yield 69.3 g ofpoly(HPMA-co-MA-GG-ONp) as a white solid. The structure was verified by¹H NMR and the product shown to be free from substantial impurities(e.g., p-nitrophenol). The amount of p-nitrophenyl ester per gram ofpolymer may be determined by UV absorbance. A wide range of copolymersbased on different monomers and/or monomer ratios may be made followingthis procedure by adjusting the stoichiometry and/or using differentmonomers.

Synthesis of poly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)BOC) and GeneralProcedure A

A solution of poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6mL) and H₂O (10 mL) was added dropwise over a 15 minute interval to asolution of tert-butyl N-(2-aminoethyl)-N-methylcarbamate (0.20 g, 1.15mmol) in water (20 mL) at 0° C. The reaction mixture was stirred at 0°C. for 15 minutes, then warmed to room temperature and stirred for 12hours. The solvents were evaporated under reduced pressure. Theresulting residue was dissolved in water (50 mL), the pH was adjusted toapproximately 8.0 with 0.1 M NaOH. The solution was filtered through aVacuCap filter, then purified using TFF (10 K). The polymer-containingsolution was washed (as part of the TFF process) with 25 mM NaClsolution (800 mL) to remove p-nitrophenol, the pH of the solution wasadjusted to approximately 4 with 0.1 M HCl, and then washed (as part ofthe TFF process) with water (400 mL). The polymer solution waslyophilized to isolate the compoundpoly(HPMA-co-MA-GFLG-NHCH₂CH₂N(Me)BOC) as a pale yellow solid (720 mg,71%).

Synthesis of Fmoc-Phe-Gly-NH—(CH₂)₆NH-Boc

To a solution of Fmoc-Phe-Gly-OH (0.66 g) in anhydrous THF (20 mL) at 0°C. under N₂ was added N,N′-dicyclohexylcarbodiimide (0.307 g) and1-hydroxybenzotriazole hydrate (0.201 g). After stirring for 15 min,N-Boc-1,6-diaminohexane (0.322 g) was added. The reaction mixture wasallowed to warm to RT and stirred overnight. Solids were filtered offand they were washed with EtOAc. The filtrate and washings were thenconcentrated under reduced pressure. The resulting residue was purifiedby flash column chromatography (0 to 10% MeOH in CH₂Cl₂) to affordFmoc-Phe-Gly-NH—(CH₂)₆NH-Boc as a white solid (0.9 g).

Synthesis of Fmoc-Phe-Gly-NH—(CH₂)₆NH₂.TFA

Fmoc-Phe-Gly-NH—(CH₂)₆NH-Boc (0.7 g) was dissolved in CH₂Cl₂ (4 mL) at0° C. under N₂ and then trifluoroacetic acid (TFA) (4 mL) was added. Thereaction mixture was allowed to warm to RT and stirred for 2 hours underN₂. The solvents were removed under reduced pressure and the residuedried at high vacuum to provide 0.71 g of Fmoc-Phe-Gly-NH(CH₂)₆—NH2.TFA.This crude material was used to prepare without further purification.

Synthesis of Fmoc-Phe-Gly-NH(CH₂)₆NH—CO-Fumagillol

To a 0° C. solution of compound Fmoc-Phe-Gly-NH(CH₂)₆—NH2.TFA (0.71 g)in anhydrous CH₂Cl₂ (20 mL) and DMF (1 mL) under N₂ was addednitrophenyl fumagill-6-yl carbonate (0.536 g). Diisopropylethylamine(DIPEA) (0.74 mL) was then added. The reaction mixture was allowed towarm to RT and then stirred overnight at the same temperature. Thesolvents were removed under reduced pressure and the resulting residuewas dissolved in EtOAc (70 mL). The EtOAc was washed with water andbrine. The ethyl acetate solution was then dried over MgSO₄, filteredand concentrated under reduced pressure. The residue was purified byflash column chromatography (0 to 10% MeOH in CH₂Cl₂) to provideFmoc-Phe-Gly-NH—(CH₂)₆NH—CO-fumagillol as an off white solid (0.81 g)

Synthesis of H-Phe-Gly-NH(CH₂)₆NH—CO-fumagillol

To a 0° C. solution of compound Fmoc-Phe-Gly-NH—(CH₂)₆NH—CO-fumagillol(0.80 g) in anhydrous CH₂Cl₂ (20 mL) under N₂ was added DBU (0.15 g).The reaction mixture was allowed to warm to RT. The solvent was removedunder reduced pressure and the resulting residue was purified by flashcolumn chromatography (0 to 10% MeOH in CH₂Cl₂) to provideH-Phe-Gly-NH—(CH₂)₆NH—CO-fumagillol as a pale yellow gum (0.45 g, 76%).

Synthesis ofpoly[HPMA-co-MA-GGFG-N-(6-aminohexyl)carbamoylfumagillol]and GeneralProcedure B

To a solution of poly(HPMA-co-MA-GG-ONp) (0.68 g) in anhydrous DMF (12mL) at 0° C. under N₂ was added H-Phe-Gly-NH(CH₂)₆NHCO-fumagillol (0.45g) in anhydrous DMF (5 mL) followed by the addition ofdiisopropylethylamine (DIPEA) (0.25 mL). The reaction mixture wasallowed to warm to RT and after stirring overnight under N₂,3-amino-1-propanol (0.032 g) was added. The mixture was allowed to stirfor an additional hour. The solvent was removed under reduced pressureand the resulting residue was dissolved in 300 mL of distilled water andextracted with EtOAc (4×). A saturated aqueous NaCl solution (50 mL) wasused to facilitate the phase separation. Traces of EtOAc were removedfrom the polymer solution by stirring under a flow of nitrogen gas. Thepolymer solution was filtered through a vacu cap filter (pH=5.56),concentrated to 30 mL by TFF with a 10K capsule and washed with water(700 mL) by TFF. The polymer was then lyophilized to provide the desiredpolymer conjugatepoly[HPMA-co-MA-GGFG-N-(6-aminohexyl)carbamoylfumagillol]as a light pinkfoam (0.685 g). The spiroepoxide content was measured by reaction with2-mercaptopyrimidine and determined to be 0.4 mmol/g.

Synthesis of poly[HPMA-co-MA-GGLG-N-(6-aminohexyl)carbamoylfumagillol]

Using standard techniques, the dipeptide H-Leu-Gly-NH(CH₂)₆NHCO-Fum wasprepared and coupled to poly(HPMA-co-MA-GG-ONp) using General ProcedureB.

Synthesis of poly[HPMA-co-MA-GGVG-N-(6-aminohexyl)carbamoylfumagillol]

Using standard techniques, the dipeptide H-Val-Gly-NH(CH₂)₆NHCO-Fum wasprepared and coupled to poly(HPMA-co-MA-GG-ONp) using General ProcedureB.

Synthesis of poly[HPMA-co-MA-GGGG-N-(6-aminohexyl)carbamoylfumagillol]

Using standard techniques, the dipeptide H-Gly-Gly-NH(CH₂)₆NHCO-Fum wasprepared and coupled to poly(HPMA-co-MA-GG-ONp) using General ProcedureB.

Synthesis ofpoly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoylfumagillol] viapoly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexylamine.HCl)]

General Procedure C was followed using cis-1,4-diaminocyclohexane (0.914g) and poly(HPMA-co-MA-GFLG-ONp) (1.5 g) to yieldpoly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexylamine.HCl)] as an off-whitesolid (1.08 g).

General procedure F was followed usingpoly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexylamine.HCl)] (0.98 g),p-nitrophenyl fumagill-6-yl carbonate (0.465 g) and DIEA (0.268 g) inDMF 16 mL. The solvent was evaporated and the solution diluted withwater. The aqueous phase (500 mL total) was extracted with ethyl acetate(80 mL total) and purified by TFF using an additional 350 mL of water.The retentate was diluted with water, extracted with ethylacetate andlyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoylfumagillol] as alight pink solid (0.79 g).

¹H NMR (DMSO-d6): δ 7.90-8.35 (m, 4H, amide-NH), 7.0-7.70 (m, 25H,Phenylalanine and amide-NH), 5.26 (m, H-5-Fum), 5.18 (bt, alkene-Fum),4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, phenylalanine alpha proton),4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H), 3.26 (s,3H, OMe-Fum), 2.80-3.10 (m, 31H), 2.17 (m, 2H, allylic-Fum), 0.37-2.0[m, 166H {1.69 (s, 3H, Fum-Me), 1.59 (s, 3H, Fum-Me), 1.07 (s, 3H,Fum-Me)}].

Synthesis of poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂.HCl) and General ProcedureC for the Reaction of Diamines with poly(HPMA-co-MA-GFLG-ONp)

A solution of ethylenediamine (0.33 g, 5.49 mmole) in water (20 mL), pH11.7, was adjusted to pH 9.1 by the addition of 37% aq HCl (17-18drops). The solution was cooled in an ice bath andpoly(HPMA-co-MA-GFLG-ONp) (1.03 g) in DMF (6 mL) was added dropwise over20 minutes while maintaining the temperature below 4° C. The solutionwas stirred 20 minutes at 4° C., 50 minutes at room temperature to givea lemon yellow solution, pH 8.1. The solution was evaporated at 40° C.H₂O (3 ×10 mL) was added and evaporated. The product was diluted withwater (60 mL), the solution adjusted with NaOH to pH 8.0. The solutionwas filtered through a VacuCap filter and purified by TFF as follows.The polymer solution was first washed with 25 mM NaCl solution (800 mL)to remove p-nitrophenol. The solution was washed with water (400 mL)then adjusted to pH 4 with 0.1 M HCl. The TFF retentate was collectedand the filter was washed with 2×10 mL of water. The combined retentateand washes gave a polymer solution which was lyophilized to isolate thecompound poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂.HCl) as a pale yellow solid(0.71 g, 72%).

Synthesis of N-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol andGeneral Procedure D

A solution of p-nitrophenyl fumagill-6-yl carbonate (400 mg, 0.89 mmol)and (R)-2-amino-3-methyl-1-butanol (280 mg, 2.71 mmol) were stirred inethanol (10 mL) at room temperature for 12 hours. The yellow solutionwas concentrated and the residue purified by flash chromatography(methanol/methylene chloride) to yieldN-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol (340 mg, 0.83mmol) as a colorless oil.

Synthesis of N-(6-hydroxyhexyl)carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (150 mg) in ethanol (10 mL) and 6-aminohexanol (48 mg). Theproduct was isolated as a colorless oil (110 mg, 78%).

Synthesis of N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (100 mg) in ethanol (3 mL) and THF (1 mL) and cycloleucinol(52 mg) to afford N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol asan oil (50 mg).

Synthesis of N-(1-hydroxy-2-methylpropan-2-yl)carbamoylfumagillol

General Procedure D was followed using p-nitrophenyl fumagill-6-ylcarbonate (100 mg) in ethanol (3 mL) and THF (2 mL) and2-amino-2-methylpropanol (40 mg) to affordN-(1-hydroxy-2-methylpropan-2-yl)carbamoylfumagillol as an oil (37 mg).

Synthesis of fumagill-6-yl(2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate

General procedure D was followed. The S-prolinol (68 mg, 0.67 mmol) wasreacted with p-nitrophenyl fumagill-6-yl carbonate (150 mg, 0.335 mmol)in ethanol (4 mL) The product was purified by flash chromatography(methanol/methylene chloride) to yield fumagill-6-yl(2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate as a white foam (81 mg,63%).

Synthesis of N-(6-aminohexyl)carbamoylfumagillol

A solution of 1,6-diaminohexane (0.13 g) in methanol (8 mL) was cooledto 0° C. and p-nitrophenyl fumagill-6-yl carbonate (0.13 g) in methanol(2 mL) was added dropwise. The solvent was reduced to about 2 mL byrotary evaporation. Ethyl acetate was added and the organic phase waswashed with water, 0.1 N NaOH, water, brine and dried with sodiumsulfate. The solvent was evaporated and the residue dissolved in ethanol(15 mL). DL-tartaric acid (16 mg) was added, the solution was storedovernight and then evaporated to about 0.5 mL. Ether was added and awhite solid formed. The solid was collected by filtration, washed withether and dried to yield the tartrate salt ofN-(6-aminohexyl)carbamoylfumagillol (74 mg).

Synthesis of Fumagill-6-yl [trans-(4-aminocyclohexyl)]carbamate

To a solution of trans-1,4-diaminocyclohexane (1.3 g) in methanol (80ml) at 0-5° C. was added over 30 min a solution of fumagill-6-yl4-nitrophenyl carbonate (1.0 g) in methanol (20 ml,) and CH₂Cl₂ (20 ml)and then stirred for 30 minutes. After concentration to 20 ml on arotavap and dilution with ethyl acetate (75 ml) the organic layer waswashed with water (30 ml), 0.1 N NaOH (30 ml), water and brine (30 ml),dried (MgSO₄) and concentrated under reduced pressure to give 0.78 g ofa solid. This was dissolved in ethanol (80 ml) and DL-tartaric acid (127mg) was added. After 1 hour a solution formed which was allowed to standovernight before being concentrated under reduced pressure to removevirtually all the ethanol MTBE (100 ml) was added and concentratedfollowed by MTBE (30 ml). The solids were collected by filtration andwashed with MTBE (2×10 ml) and dried under vacuum to give fumagill-6-yl[trans-(4-aminocyclohexyl)]carbamate hemi-tartrate (0.73 g); m.p.180-185° C.

Synthesis of poly[HPMA-co-MA-GFLG-NH(CH₂)₆NH₂.HCl]

General Procedure C was followed using 1,6-diaminohexane (621 mg, 5.36mmol) and poly(HPMA-co-MA-GFLG-ONp) (1.0 g). The crude product waspurified by TFF (5 K) using aqueous NaCl (25 mM) and then acidified topH 4.0 with 0.1 M HCl and further purified by TFF with water to yieldpoly[HPMA-co-MA-GFLG-NH(CH₂)₆NH₂.HCl] as an off-white solid (860 mg).

Synthesis of p-nitrophenylN-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagill-6-yl carbonate andGeneral Procedure E

To a solution of the alcoholN-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol (1.11 g) inmethylene chloride at 0° C. under N₂ was added DMAP (660 mg, 5.40 mmol)followed by the portionwise addition of p-nitrophenyl chloroformate (810mg). The reaction mixture was stirred at 0° C. for 1 hour. The solventwas evaporated and the resulting residue was dissolved in EtOAc andwashed with water, brine and dried (Na₂SO₄). Evaporation of EtOAcprovided the crude product, which was purified by flash chromatography(silica, eluting with 100% hexanes and then with 2-30% EtOAc). Thefractions containing pure product were combined and evaporated toisolateN-[(2R)1-(p-nitrophenolcarbonylhydroxy-2-methylbutan-2-yl]carbamoylfumagillol(1.25 g, 80%) as a white solid.

Synthesis ofN-[1-(p-nitrophenoxycarbonylhydroxymethyl)-2-methylpropan-2-yl)carbamoylfumagillol

Following General Procedure E, dimethylalcohol (60 mg), p-nitrophenylfumagill-6-yl carbonate (46 mg), and DMAP (37 mg) were reacted inmethylene chloride (8 mL). The reaction mixture was diluted with ethylacetate and washed with water (3×) and then brine. The organic phase wasdried (Na₂SO₄) and evaporated to a yellow foam (87 mg) which was usedwithout further purification.

Synthesis ofN-[1-(p-nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol

Following General Procedure E,N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol (product fromExample 14, 74 mg), p-nitrophenyl chloroformate (53 mg), and DMAP (43mg) were reacted in methylene chloride (5 mL). After the extractiveworkup,N-[1-(p-nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol(100 mg) was used without further purification.

Synthesis ofpoly[HPMA-co-MA-GFLG-NH(CH₂)₆NHcarbamoyl-[1-hydroxy-3-methylbutan-2-yl]carbamoylfumagillol]and General Procedure F

To a solution of polymer (400 mg) and p-nitrophenylN-[(2R)1-hydroxy-3-methylbutan-2-yl]carbamoylfumagill-6-yl carbonate(240 mg) in DMF (8 mL) at 0° C. was added DIEA (0.11 g) dropwise. Thesolution was stirred at 0° C. for one hour and allowed to warm to roomtemperature. After 3 days, the solvent was evaporated and water (80 mL)was added. The aqueous phase was extracted with ethyl acetate (500 mLtotal) until none of the starting carbonate was detectable by MS. Theaqueous phase was purified by TFF (10 K) and the retentate lyophilizedto yield the conjugate as a white solid (380 mg, 77%).

¹H NMR (DMSO-d6): δ 8.25 (bs, 2H, amide-NH), 8.0 (bs, 1H, amide-NH),7.70 (bs, 2H, amide-NH), 7.10-7.30 (m, 15H, Phenylalanine and amide-NH),7.10 (bt, 1H, NH-Fum), 6.92 (bd, 1H, NH-Fum), 5.26 (m, H-5-Fum), 5.18(bt, alkene-Fum), 4.50-4.80 (m, 1H, phenylalanine alpha proton),4.0-4.21 (m, 1H, leucine alpha proton), 3.50-3.84 (m, 19H), 3.29 (s, 3H,OMe-Fum), 2.80-3.10 (m, 28H), 2.51 (d, 1H, J=4.4 Hz, H-2-Fum), 2.19 (m,2H, allylic-Fum), 0.82-1.92 [m, 131H {1.84 (m, 2H, Fum), 1.72 (s, 3H,Fum-Me), 1.60 (s, 3H, Fum-Me), 1.09 (s, 3H, Fum-Me), 0.84 (dd, 6H,Fum-isopropyl}].

Synthesis of poly[HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoylfumagillol]

General procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂.HCl) (200 mg), p-nitrophenylfumagill-6-yl carbonate (100 mg) and DIEA (57 mg) in DMF (10 mL). Theproduct was purified by TFF (10 K) with water and lyophilized to yieldthe conjugate as a pale yellow solid (160 mg).

Synthesis ofpoly[HPMA-co-MA-GFLG-N(Me)-(2-methylaminoethyl)carbamoylfumagillol]

General procedure F was followed usingpoly(HPMA-co-MA-GFLG-N(Me)CH₂CH₂NHMe.HCl) (200 mg), p-nitrophenylfumagill-6-yl carbonate (100 mg) and DIEA (57 mg) in DMF (5 mL). Theproduct was purified using TFF (10 K) with water and lyophilized toyield the conjugate as an off-white solid (180 mg).

Synthesis ofpoly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagillol

General procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂.HCl) (200 mg), p-nitrophenyldihydrofumagill-6-yl carbonate (200 mg) and DIEA (57 mg) in DMF (10 mL).The product was purified by TFF (10 K) with water (150 mL) andlyophilized to yieldpoly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagillol as apale yellow solid (160 mg).

Synthesis of poly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol]

General procedure F was followed using poly(HPMA-co-MA-GFLG-NHCH₂CH₂CH₂NH₂.HCl) (220 mg), p-nitrophenyl fumagill-6-yl carbonate (110 mg) andDIEA (63 mg) in DMF (6 mL). The solvent was evaporated and the resultingsolution diluted with water. The aqueous phase was extracted with ethylacetate and purified by TFF using 350 mL of water. The retentate waslyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol] as a lightpink powder (200 mg).

Synthesis of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol]

General procedure F was followed usingpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexylamine.HCl] (1.0 g),p-nitrophenyl fumagill-6-yl carbonate (0.48 g) and DIEA (0.27 g) in DMF(25 mL). The solvent was evaporated and the solution diluted with water.The aqueous phase (300 mL) was extracted with ethyl acetate (700 mLtotal) and purified by TFF using an additional 350 mL of water. Theretentate was lyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(4-aminocyclohexyl)carbamoylfumagillol] as alight pink solid (0.9 g).

¹H NMR (DMSO-d6): δ 8.10-8.35 (m, 3H, amide-NH), 7.90-8.10 (m,amide-NH), 7.05-7.32 (m, 22H, amide-NH) 5.27 (m, H-5-Fum), 5.18 (bt,alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, phenylalanine alphaproton), 4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H),3.27 (s, 3H, OMe-Fum), 2.80-3.20 (m, 33H), 2.56 (d, 1H, H=3.90 Hz,H-2-Fum), 2.18 (m, 2H, allylic-Fum), 0.37-2.0 [m, 147H {1.70 (s, 3H,Fum-Me), 1.60 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum-Me)}].

Synthesis ofpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexyl)carbamoylfumagillol]

General procedure F was followed usingpoly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexylamine.HCl)] (1.0 g),p-nitrophenyl fumagill-6-yl carbonate (0.48 g) and DIEA (0.27 g) in DMF25 mL. The solvent was evaporated and the solution diluted with water.The aqueous phase (300 mL) was extracted with ethyl acetate (700 mLtotal) and purified by TFF using an additional 350 mL of water. Theretentate was lyophilized to yieldpoly[HPMA-co-MA-GFLG-N-(3-aminohexyl)carbamoylfumagillol] as a lightpink solid (0.9 g).

¹H NMR (DMSO-d6): δ 7.90-8.35 (m, 4H, amide-NH), 7.0-7.70 (m, 25H,Phenylalanine and amide-NH), 5.26 (m, H-5-Fum), 5.18 (bt, alkene-Fum),4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, phenylalanine alpha proton),4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H), 3.26 (s,3H, OMe-Fum), 2.80-3.10 (m, 31H), 2.17 (m, 2H, allylic-Fum), 0.37-2.0[m, 166H {1.69 (s, 3H, Fum-Me), 1.59 (s, 3H, Fum-Me), 1.07 (s, 3H,Fum-Me)}].

Synthesis ofpoly[HPMA-co-MA-GFLG-N-[2-(4-aminophenyl)ethyl]carbamoylfumagillol]

To a suspension of poly[HPMA-co-MA-GFLG-OH] (200 mg),N42-(4-aminophenyl)ethyl]carbamoylfumagillol] (100 mg) and DIEA (75 mg)in DMF (6 mL) at 0° C. was added EDCI (total 44 mg) in portions. Thesolution was allowed to warm to room temperature and stirred overnight.The solvent was evaporated, the residue was suspended in water and thesuspension extracted with EtOAc (7 times, total 250 mL). The aqueousphase was purified by TFF (10 K) using water (350 mL). The retentate waslyophilized to afford the polymer as a white fluffy solid (170 mg).

Synthesis ofpoly[HPMA-co-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoylfumagillol]

To a solution of 2,2′-(Ethylenedioxy)bis(ethylamine) (0.79 g, 5.34 mmol)in distilled water (20 mL) at 0° C. (pH=11.56) was added conc. HCl untilpH of the solution was 9.01 (measured by pH meter).Poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 mL) and H₂O (10mL) was added to the amine-containing solution dropwise over a period of15 minutes and the reaction mixture was stirred at 0° C. for 15 minutes.The reaction mixture was then allowed to warm to room temperature andstirred for 2 hours. The pH of the solution was measured to be 8.15. Thereaction mixture was diluted with distilled water (300 mL) and filteredthrough a VacuCap filter, reaction flask was washed with water (100 mL).The polymer solution was concentrated to 40 mL by TFF (10 K) and waswashed with 25 mM NaCl (800 mL) to remove p-nitrophenol, the pH was thenadjusted to 4 with 0.1 M HCl and finally washed with water (400 mL). Thepure polymer solution was lyophilized to isolatepoly[HPMA-co-MA-GFLG-NH-2-[2-(2-aminoethoxy)ethoxy]ethylamine.HCl] as apink solid (800 mg, 78%).

To a mixture of p-nitrophenyl fumagill-6-yl carbonate (93 mg, 0.208mmol) andpoly[HPMA-co-MA-GFLG-N-2-[(2-(2-aminoethoxy)]ethoxy)ethylamine.HCl] (200mg, 0.104 mmol) in anhydrous DMF (5 mL) at 0° C. under N₂ was added DIEA(57 mg, 0.416 mmol). The reaction mixture was allowed to warm to roomtemperature and stirred for 12 hours. The solvent was removed underreduced pressure and the resulting residue was suspended in water (30mL) and extracted with EtOAc (aqueous and organic phases from theemulsion formed were separated using centrifuge) to remove excess ofp-nitrophenyl fumagill-6-yl carbonate and p-nitrophenol. Nitrogen waspassed through the aqueous solution to remove traces of EtOAc and it waspurified using TFF (5K) by washing it with water (150 mL) to remove DIEAhydrochloride. The polymer solution was lyophilized to obtain thedesired polymer conjugatepoly[HPMA-co-MA-GFLG-N-2-[2-(2-aminoethoxy)ethoxyethyl]carbamoylfumagillol](220 mg, 95%) as an off-white solid.

Synthesis of poly[HPMA-co-MA-GFLG-NH-(6-aminodecyl)carbamoylfumagillol]

To a mixture of p-nitrophenyl fumagill-6-yl carbonate (300 mg, 0.67mmol) and poly[HPMA-co-MA-GFLG-N-10-[decylamine.HCl] (300 mg, 0.15 mmol;made in a similar manner to Example 33 except 1,10-diaminodecane wasused as the amine) in anhydrous DMF (6 mL) at 0° C. under N₂ was addedDIEA (83 mg, 0.64 mmol). The reaction mixture was allowed to warm toroom temperature and stirred for 12 hours. The solvent was removed underreduced pressure and the resulting residue was suspended in water (30mL) and extracted with EtOAc (aqueous and organic phases from theemulsion formed were separated using a centrifuge) to remove excess ofp-nitrophenyl fumagill-6-yl carbonate and p-nitrophenol. Nitrogen waspassed through the aqueous solution to remove traces of EtOAc. The crudeaqueous solution was purified using TFF (10K) by washing with water (150mL) to remove DIEA hydrochloride. The polymer solution was lyophilizedto obtain the desired polymer conjugatepoly[HPMA-co-MA-GFLG-NH-(10-aminodecyl)carbamoylfumagillol] (300 mg,87%) as an off-white solid.

Synthesis of N-(2-acetamidoethyl)carbamoylfumagillol

To a solution of p-nitrophenyl fumagill-6-yl carbonate (200 mg) inethanol (5 mL) at 0° C. was added N-(2-aminoethyl)acetamide (0.132 mL).The solution was stirred at 0° C. for one hour and overnight at roomtemperature. The reaction was diluted with ethyl acetate, washed withwater. The aqueous phase was back extracted with ethyl acetate and thecombined organic phases dried (MgSO₄). The crude product was purified byflash chromatography. The product was a yellow solid (120 mg).

Synthesis of the Following Compound

To a solution of poly(HPMA-co-MA-GFLG-NHCH₂CH₂NH₂.HCl) (200 mg) andN-(5-carboxypentyl)carbamoylfumagillol (96 mg) in DMF (6 mL) at 0° C.was added DIEA (104 mg) followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (42 mg).The solution was allowed to warm to RT and stirred overnight. Thesolvent was evaporated and the residue dissolved in water (50 mL) andextracted with ethyl acetate (200 mL). The aqueous phase was purified byTFF with water (450 mL). The retentate was lyophilized to yield thepolymer (200 mg) as a pale yellow solid.

Synthesis of the Following Compound

To a solution of poly[HPMA-co-MA-GFLG-N(CH₂)₆NH₂.HCl] (216 mg),2-carboxyethylcarbamoylfumagillol (91 mg) in DMF (8 mL) at 0° C. wasadded DIEA (118 mg) followed byN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (88 mg).The solution was allowed to warm to room temperature and stirredovernight. The solvent was evaporated and the residue dissolved in water(50 mL) and extracted with ethyl acetate (200 mL). The aqueous phase waspurified by TFF (10 K) with water (1 L). The retentate was lyophilizedto yield the polymer (170 mg) as a pale yellow solid.

Synthesis of the Following Compound

General Procedure F was followed usingpoly(HPMA-co-MA-GFLG-NHCH₂CH₂CH₂NH₂.HCl) (220 mg) and carbonate (Example24, 100 mg) in DMF (6 mL) with DIEA (63 mg). The reaction was extractedwith ethyl acetate. Following TFF (10 K) purification with water, andlyophilization, the product was isolated as a light pink powder (140mg).

BocNHCH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl (Alkylation of N—BOC,N′-methylethylenediamine with chloroacetylcarbamoylfumagillol)

A solution of TNP-470 (0.2 g) and DIEA (0.105 g) in DMF (3 mL) wascooled to 0° C. A solution of tert-butylN-[2-(methylamino)ethyl]carbamate (0.105 g) in DMF (3 mL) was added, andthe mixture was stirred for 3 hours at 0° C. and then overnight. Thereaction was diluted with ethyl acetate and extracted with water. Theaqueous phase was back extracted with ethyl acetate, and the combinedorganic phases were extracted with brine, dried (MgSO₄) and evaporatedto afford an oil. Purification by silica gel chromatography(methanol/methylene chloride) and evaporation of the product fractionsgave BocNHCH₂CH₂N(Me)CH₂C(O)NHC(O)₂-fumagill-6-yl a white foam (0.16 g,60%).

Reaction of tert-butyl N-[2-aminoethyl]carbamate withchloroacetylcarbamoylfumagillol

A 30 uL aliquot of a 1 M solution of Boc-ethylenediamine in DMF wasadded to DMF (270 uL). The solution was cooled to 0° C., and a solutionof TNP-470 (48 mg) in DMF (600 uL) was added dropwise over 2 minutes.The reaction was monitored by LC/MS. The largest amount of the desiredalkylation product observed was 34%. Carbamoylfumagillol was alsoproduced. The ratio of desired product to carbamoylfumagillol was 1.0 to0.4. Attempted isolation of the desired product resulted in theisolation of hydantoin and fumagillol. Thus, the desired product couldnot be isolated because of the rate of decomposition. Thus TNP-470 couldnot be alkylated according to the described method.

In vivo testing DIO C57Bl6 Mice—Weight Changes, Food Consumption, BodyComposition

C57Bl6 male mice (N=6) thirteen weeks old with an average of weight of34 g were ad libitum fed TD.06414 a high fat diet composed of 60% Kcalfrom fat (Harlan diet). On study day 1 animals were randomized intogroups so that the average weight of each group was 33.9 g. The micewere treated with either phosphate buffered saline (vehicle), TNP-470,or Compound 16 (dorsal, subcutaneous administration). Treatment wascontinued for 31 days at the doses and on the schedule shown in theTable below. Animals were weighed every other day. Food consumption wasmeasured weekly. On day 33 gross pathology was performed to determinebody composition.

FIG. 1 compares the weight loss in an obese DIO mouse followingtreatment with a fumagillol-conjugate compound of the present invention(Compound 16) or TNP-470 (synthetic fumagillin analog) at varyingdoses/regimens as recited in Table 2.

TABLE 2 Body Weight Dose Day 33 mg/kg/day Dose* Schedule Drug 39.6 0 0qod Vehicle 34.1 0.5 1.12 qod TNP-470 30.4 0.5 0.18 qod Compound 16 27.41.5 0.56 q4d Compound 16 *Daily Dose as micromoles/kg Fumagillol

The results in FIG. 1 show an increase in weight in the vehicle controlgroup of 16% and a decrease in weight of 19% when administering Compound16 in a q4d dosing regimen. Administration of compound 16 provides boththerapeutic and prophylactic effects. Specifically, compound 16 inducesor increases weight loss and it also prevents an increase in weight.Compound 16 is superior to TNP-470 in the degree of weight loss.Compound 16 is superior to TNP-470 in that fumagillol doses are reduced.

Table 3 compares the body fat composition in an obese DIO mousefollowing treatment with Compound 16 or TNP-470 at the varyingdoses/regimens described herein. Analysis was performed at Day 33 grosspathology. Total fat as a percentage of BW in the vehicle group was13.2% while total fat in the groups treated with Compound 16 was 8.2% (1mg/kg qod) or 5.6% (6 mg/kg q4d).

TABLE 3 Weights in grams, group averages Total Epididymal InguinalRetroperitoneal B.W. Fat Fat Fat Fat Liver Vehicle 39.6 5.24 2.21 1.931.10 1.46 TNP qod 34.1 3.55 1.54 1.21 0.80 1.15 Cmpd 16 30.4 2.52 1.180.92 0.42 1.10 1 mg/kg qod Cmpd 16 27.4 1.55 0.78 0.53 0.23 1.17 6 mg/kgq4d

FIG. 2 compares the average daily food consumption in an obese DIO mousefollowing treatment with Compound 16 or TNP-470 at varyingdoses/regimens. The results in FIG. 2 show decrease food consumptionfollowing treatment with Compound 16 and Compound 16 causes a greaterreduction in food consumption than TNP-470.

FIG. 3 compares the body composition (fat v. body weight) in an obeseDIO mouse following treatment with Compound 16 or TNP-470 at varyingdoses/regimens. The results in FIG. 3 show that the amount of bodyweight lost is directly correlated to fat loss.

In Vivo Testing DIO C57Bl6 Mice—Weight Changes, Food Consumption,Glucose Tolerance and Body Composition Dose Response

C57Bl6 male mice (N=6) fifteen weeks old with an average of weight of 42g were ad libitum fed TD.06414 a high fat diet composed of 60% Kcal fromfat (Harlan diet). On study day 1 animals were treated with eitherphosphate buffered saline (vehicle), or Compound 16 at various doses(dorsal, subcutaneous administration). Treatment was continued for 29days at the doses and on the schedule shown in the Table below. Animalswere weighed every other day. Food consumption was measured weekly. Onday 24 (mice had been most recently treated with Compound 16 on day 21)an overnight fasted IP-Glucose Tolerance Test (GTT) was administered tothe vehicle group and the four Compound 16 treatment groups. Each animalwas weighed and a baseline fasted glucose measurement collected. Eachanimal was given a 1 gram per kilogram dose of dextrose as a 25%solution by intraperitoneal injection. Blood glucose levels weremeasured at 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120minutes after IP administration of glucose (via tail vein blood samplesby using an AlphaTRAK blood glucose monitoring system (including glucosemeter and test strips) from Abbott Laboratories, (North Chicago, Ill.,USA). The AlphaTRAK meter displays results between 20 and 750 mg/dL(1.1-41.7 mmol/L). On day 32 (mice had been most recently dosed on day29) animals were fasted for three hours, weighed, blood collected bycardiac puncture and gross pathology was performed to determine bodycomposition. Blood analysis was performed by Idexx laboratories. Bloodglucose was 278, 290, 265, 259 and 227 mg/dL for doses of 0, 0.2, 0.6,2.0 and 6.0 respectively. BUN was 21.8, 22.0, 19.7, 15.3, and 16.5 fordoses of 0, 0.2, 0.6, 2.0 and 6.0 respectively.

Table 4 shows the blood glucose level as a function of time and the doseof Compound 16. Table 4 shows that higher doses of Compound 16 result inlower blood glucose levels even at the lowest dose of 0.2 mg/kg; theseresults are displayed in FIG. 4.

4 Blood Glucose, Group averages Dose Average Blood Glucose mg/dL groupaverage ± SEM mg/kg BW (g) Fasting 15 min 30 min 60 min 90 min 120Vehicle - 0 43.0 226 ± 22.9 502 ± 30.3  621 ± 38.4*  580 ± 48.7* 459 ±54.0 297 ± 33.7 0.2 43.4 254 ± 5.8  505 ± 33.9 446 ± 20.5 456 ± 25.9 341± 15.5 241 ± 16.8 0.6 40.9 206 ± 14.9 442 ± 50.0  441 ± 35.54 388 ± 56.7302 ± 43.3 192 ± 12.9 2.0 36.8 198 ± 11.6 388 ± 20.7 361 ± 24.3 327 ±26.7 244 ± 24.0 166 ± 6.4  6.0 33.0 185 ± 6.5  473 ± 39.1 355 ± 23.8 324± 19.1 240 ± 6.7  177 ± 9.3  *one mouse in each group had bloodglucose >750 mg/dL; treated as equal to 750 mg/dL

Table 5 shows that increasing doses of Compound 16 result in significantincreases in weight loss at doses greater than 0.2 mg/kg q4d. Table 6shows that doses of 2 mg/kg q4d and 6 mg/kg q4d were associated withsignificant reductions in food consumption relative to vehicle controlsand that food consumption is dose-responsive. From day 9-29, the weeklyfood consumption in the 2 mg/kg group was 90% of vehicle while the foodconsumption in the 6 mg/kg group was 75% of vehicle.

TABLE 5 Body Weights Day 32 Dose Group BW (g) ± Weight Change mg/kg SEMfrom day 1 Fumagillol* Vehicle-0 45.7 ± 1.12 +8.3% 0.00 0.2 46.5 ± 0.65+10.5% 0.02 0.6 43.3 ± 0.42 +2.9% 0.06 2.0 38.8 ± 0.70 −7.4% 0.18 6.033.6 ± 0.72 −20.0% 0.54 *micromoles/kg/day; dosing q4d

TABLE 6 Weekly food consumption, Group Averages Day 1-8 Day 9-15 Day15-22 Day 22-29 Group Food (g) Food (g) Food (g) Food (g) Vehicle 22.423.3 22.4 21.5 0.2 mg/kg 25.8 22.6 20.3 19.7 0.6 mg/kg 19.5 21.5 20.218.4   2 mg/kg 14.3 22.2 19.9 18.5   6 mg/kg 9.0 17.5 17.8 16.1

Table 7 shows that adipose tissue is lost in preference to other tissuesas the mice in the control group are about 13% fat while the mice in the2 mg/kg q4d group and 6 mg/kg q4d group are 11% and 10% fat,respectively.

TABLE 7 Tissue weights day 32, group averages avg avg Tissue WeightsAverages (g) Dose B.W. Total Epid. Ing. RP Fat % mg/kg (g) Fat Fat FatFat Liver BW 0.0 45.70 5.88 1.88 2.65 1.35 2.00 12.9% 0.2 46.50 6.022.01 2.54 1.48 2.06 13.0% 0.6 43.32 6.01 2.24 2.51 1.26 1.75 13.9% 2.038.82 4.42 1.72 1.81 0.89 1.44 11.4% 6.0 33.60 3.25 1.11 1.54 0.60 1.309.7%

The results in FIG. 5 show increasing weight loss following treatmentwith Compound 16 at doses greater than or equal to 0.6 mg/kg utilizing aq4d schedule. The weight loss is dose-responsive, higher doses causegreater weight loss.

The results in Table 8 show reductions in cholesterol, triglycerides,HDL, LDL and HDL/LDL ratios associated with increasing doses of Compound16. These results are displayed in FIG. 6.

TABLE 8 Lipids in blood day 32 HDL Dose CHOLESTEROL TRIGLYCERIDECHOLESTEROL LDL HDL/LDL mg/kg mg/dL mg/dL mg/dL mg/dL ratio 0  244 ±12.6 140 ± 7.7 112 ± 4.0 24 ± 2.2 4.8 ± 0.4 0.2 253 ± 8.4  158 ± 10.9117 ± 2.7 24 ± 1.1 4.9 ± 0.2 0.6 210 ± 3.7 116 ± 3.1 106 ± 1.2 20 ± 0.35.4 ± 0.1 2 149 ± 4.7  95 ± 8.7  92 ± 2.6 10 ± 0.5 9.0 ± 0.4 6 109 ± 2.6 81 ± 13.6  72 ± 1.3 8 ± 0.5 9.0 ± 0.5 Values are ± SEM

The results in Table 9 show favorable changes in alkaline phosphatase,SGPT, SGOT and CPK associated with increasing doses of Compound 16.

TABLE 9 Blood enzyme analysis Dose ALKALINE SGPT SGOT mg/kg PHOSPHATASE(ALT) (AST) CPK 0 61 ± 1.2  74 ± 11.2 72 ± 5.4 34 ± 2.5  0.2 51 ± 3.2 79 ± 16.8  77 ± 10.1 74 ± 18.5 0.6 44 ± 1.4 50 ± 2.3 57 ± 2.4 66 ± 11.32 43 ± 2.4 35 ± 3.1 53 ± 3.7 68 ± 17.8 6 36 ± 0.9 42 ± 5.9 59 ± 2.2 84 ±13.3

Example

C57Bl6 male mice (N=6) fifteen weeks old with an average of weight of 42g were ad libitum fed TD.06414 a high fat diet composed of 60% Kcal fromfat (Harlan diet). On study day 1 animals were treated on a q4d schedulewith either phosphate buffered saline (vehicle), or Compounds 16, 28,29, or 30 at doses of 2 mg/kg or Compound 31 at 6 mg/kg (dorsal,subcutaneous administration). Animals were weighed every other day. FIG.10 compares the weight loss in an obese DIO mouse following treatmentfor 23 days with various conjugates of the present invention. Theresults in FIG. 10 show that changes in the linker alone result inchanges in the degree of weight loss.

Example

Sprague Dawley male rats (N=3) nine to ten weeks old with an averageweight of 300 g were ad libitum fed standard rodent diet (PharmaServ labdiet 5001). FIG. 7—Rats were treated with Compound 16 at either 100mg/kg or 200 mg/kg (IV, tail vein) on days 1, 8, 15, 22 and 29. The ratswere weighed periodically and bled on day 10, day 17, day 24. Forin-life blood collections, the rats were anesthetized with an inhalationmixture of 4% Isoflurane and 1.5% oxygen, then blood was collected viaretro-orbital plexus puncture at a volume of at least 1 mL. On day 31animals were weighed, blood collected by cardiac puncture and grosspathology was performed to determine body composition. Within the limitsof comparison to normal ranges and pre-dose data, clinical findings foralbumin, albumin/globulin ratio, alkaline, phosphatase, ALT (SGPT), AST(SGOT), bicarbonate, direct bilirubin, indirect bilirubin, totalbilirubin, BUN, BUN/creatinine ratio, calcium, chloride, cholesterol,CK, creatinine, globulin, glucose, phosphorus, potassium, sodium,sodium/potassium ratio, total protein were unremarkable. Beyond weightloss and the other findings reported here, animals appeared to begrossly normal and did not exhibit any evidence of neurotoxicity such asataxis, disorientation, tremor or convulsion. The results in FIG. 7 showthat Compound 16 is tolerated at high doses on a q7d dosing schedule.

Example

Sprague Dawley male rats (N=3, average weight 350 g) were dosed by asingle IV bolus with either vehicle, Compound 1 (30 mg/kg) or Compound16 (200 mg/kg). Blood samples were collected via saphenous vein punctureat 0, 0.25, 0.5, 1, 2, 4, 8, 24 and 48 hours. An aliquot of each samplewas diluted with methanol containing propranolol as an internal standardand analyzed by LC/MS/MS with a lower limit of quantitation of 2.5 nM.In the case of administering either Compound 1 or Compound 16, theanalyte was Compound 1. The half-life of the small molecule Compound 1is in the range of 10-15 minutes; Cmax is approximately 15 μM and occursat T₀. For the polymer conjugate, Compound 16, the released smallmolecule exhibits a Cmax of approximately 0.3 μM at about 3 hours and aterminal elimination half-life of 10 hours. These results are displayedin FIG. 9.

Example In vivo Testing DIO Levin Rats—Weight Changes, Food Consumption,Body Composition, Schedule-Dose Response, Leptin Levels

A study was conducted to evaluate the relative efficacy of thefumagillol polymer conjugate, Compound 16, and the small moleculefumagillol derivatives, Compound 1, and CKD-732 (also known as beloraniband ZGN-433). CKD-732 as referred to herein is the hemitartrate salt ofthe following structure:

Compound 1 was also tested in the form of the hemitartrate salt.

Test articles were administered subcutaneously on an every 4 day (q4d)schedule in a diet-induced obesity (DIO) Levin-DS rat model. Theefficacy of Compound 16 was also evaluated on a weekly (q7d) dosingschedule. A dietary intervention (Standard Chow, Labdiet 5001; 3.4kcal/g) was included to compare to the drug interventions. Upon arrivalat three weeks of age, the male rats received ad libitum pellets ofHarlan Diet TD.06414, 60% of calories from fat, 21% of calories fromcarbohydrate; 5.1 kcal/g. Prior to dosing the rats were randomized intogroups of three animals with an average body weight of 595 gm. The ratswere treated with either phosphate buffered saline (vehicle), Compound16 or Compound 1 or CKD-732 (dorsal, subcutaneous administration).Compound 16 was dissolved in vehicle. Compound 1 in the form of thehemitartrate and CKD-732 in the form of the hemitartrate were dissolvedin ethanol prior to dilution with vehicle. All doses were administeredat a volume of 5.0 ml/kg. Treatment was continued for 68 days at thedoses and on the schedule shown in Table 10 below. Since the molecularweight of CKD-732 is 15% greater than the molecular weight of Compound1, CKD-732 was dosed at 1.15 mg/kg while Compound 1 was dosed at 1 mg/kgfor the purpose of comparison on a molar basis. On day 1, first dose,the rats were 14 weeks of age. Also on day 1, Group 2 was switched fromthe high fat diet to a standard chow diet; the remaining groups weremaintained on a high fat diet for the duration of the study.

TABLE 10 Dose Group Test # of Dose Volume Conc. Dose # Article ratsRoute Diet Frequency (ml/kg) (mg/ml) (mg/kg) 1 Vehicle 3 SC 60% Fatq4day 5.0 0 0 2 Vehicle 3 SC Standard q4day 5.0 0 0 3 Cmpd 16 3 SC 60%Fat q4day 5.0 0.067 0.3 4 Cmpd 16 3 SC 60% Fat q4day 5.0 0.2 1.0 5 Cmpd16 3 SC 60% Fat q4day 5.0 0.6 3.0 6 Cmpd 16 3 SC 60% Fat q7day 5.0 1.26.0 7 Cmpd 1 3 SC 60% Fat q4day 5.0 0.2 1.0 8 Cmpd 1 3 SC 60% Fat q4day5.0 0.6 3.0 9 CKD-732 3 SC 60% Fat q4day 5.0 0.23 1.15

Animals were weighed every other day. Food consumption was measuredweekly. On an approximately weekly basis throughout the study, bloodsamples were taken for assessment of serum chemistries including glucoseand insulin. On Day 48 all rats underwent a 4 hour fasted Oral GlucoseTolerance Test (OGTT). Animals were dosed orally (per os, PO) at 8 mL/kg25% glucose (2 g/kg). On day 68, gross pathology was performed todetermine body composition.

FIG. 11 shows change in body weight versus study day for each group.Significant reduction in body weight was seen in both the Q4D and Q7Dpolymer conjugate dosed groups. Treatment with Compound 16 at 3 mg/kg(Q4D) or 6 mg/kg (Q7D) showed greater weight loss than the change to astandard chow. At the end of the study, Compound 16 at 3 mg/kg on a Q4Dschedule showed a 22.1% lower body weight than the vehicle control, anda 6.2% lower body weight than rats on the standard chow diet. Afterabout ten weeks, treatment with Compound 16 at 6 mg/kg on a Q7D scheduleshowed comparable body weight to treatment at 3 mg/kg dose on a Q4Dschedule. Compound 1 dosed at 1 mg/kg and CDK-732 dosed at 1.15 mg/kg ona Q4D schedule showed 3.9% or 3.2% lower body weights than vehicle.Compound 1 dosed at 3 mg/kg on a Q4D schedule showed 8.9% lower bodyweight than vehicle. The polymer conjugates are approximately ⅙^(th) ofthe active fumagillol derivative by weight.

FIG. 12 shows day 68 final body weight for all groups as a function ofthe average daily fumagillol exposure. Both Vehicle and Standard Diethad no fumagillol exposure. Compound 16 shows greater weight loss withsignificantly lower fumagillol exposure as compared to Compound 1 orCKD-732 on the same schedule as the polymer conjugate. All groups weredosed on a Q4D schedule, except for Compound 16 at 6 mg/kg, which wasdosed Q7D.

TABLE 11 Avg (n = 3) DIO Rat Final BW (g) as a function of fumagilloldose Weight Loss vs. Exposure After 68 Days on Intervention μMFumagillol Final BW Group Dose (mg/kg) Schedule kg/day (g) Vehicle HF 0q4d 0 742.3 Standard Diet 0 q4d 0 645.0 Cmpd 16 0.3 q4d 25 727.3 Cmpd 161.0 q4d 83 668.6 Cmpd 16 3.0 q4d 250 604.7 Cmpd 16 6.0 q7d 286 596.7Cmpd 1 1.0 q4d 500 717.3 Cmpd 1 3.0 q4d 1500 684.0 CKD-732 1.15 q4d 500714.0

FIG. 13 shows the reductions in serum insulin levels in male Levin DIORats maintained on a 60% fat diet and dosed with the compounds of thepresent invention on a q4d (3 mg/kg) and q7d (6 mg/kg) schedule comparedto a standard diet intervention and the vehicle group.

TABLE 12 Avg (n = 3) DIO Rat Insulin as a function of time Avg. Insulin(ng/mL) in Male Levin DIO Rats Pre-dosing Day 7 Day 15 Day 23 Day 48Vehicle 1.74 2.66 3.37 3.15 3.00 Standad diet 1.96 1.68 1.40 1.78 1.51Cmpd 16, 0.3 mg/kg 1.76 2.27 1.65 1.66 1.70 Cmpd 16, 0.6 mg/kd 2.95 2.091.38 1.72 1.91 Cmpd 16 3 mg/kg 1.49 0.66 0.54 1.26 1.08 Cmpd 16 6 mg,q7d 3.06 1.96 1.00 1.11 1.32 Cmpd 1, 1 mg/kg 1.92 1.83 1.39 1.20 1.75Cmpd 1, 3 mg/kg 2.10 1.35 1.68 1.27 1.40 CKD-732, 1.15 mg/kg 2.58 2.081.91 1.60 1.56

Table 12 shows the changes in fasting insulin levels for each group. Allgroups (except for the Vehicle control group) showed reduced insulinlevels, demonstrating that the Compounds of the present invention lowerinsulin levels on an infrequent dosing schedule.

FIG. 14 shows the results of an oral glucose tolerance test (OGTT) oninsulin levels in rats treated with the compounds of the presentinvention on a q4d and q7d schedule compared to the standard dietintervention and the vehicle groups. The standard diet intervention alsoresulted in lower insulin levels. Insulin levels remained reduced versusvehicle, in the presence of abnormally high glucose levels indicatingthat lower levels of insulin were needed to reduce blood glucose (seeFIG. 15) suggesting improved/restored insulin sensitivity.

FIG. 15 shows reduced glucose levels versus time for various treatmentsfollowing an oral glucose challenge.

TABLE 13 Blood Glucose Levels, following an oral glucose tolerance test(OGTT) in DIO Rats on a 60% high fat diet. Blood Glucose (mg/dl) in MaleLevin Rats During a Glucose Challenge Time pre- BW Group OGTT 15 30 6090 120 (g) Vehicle HF 126 158 153 163 160 132 702 Standard Diet 110 147145 134 124 129 617 Cmpd 16, 0.3 mg/kg 121 156 145 157 149 147 697 Cmpd16, 0.6 mg/kd 121 150 148 149 143 150 662 Cmpd 16 3 mg/kg 120 157 144140 127 129 582 Cmpd 16 6 mg, q7d 128 161 141 145 145 138 604 Cmpd 1, 1mg/kg 124 156 142 152 151 142 686 Cmpd 1, 3 mg/kg 123 175 171 149 147129 668 CKD-732, 123 174 171 158 153 144 686 1.15 mg/kg

FIG. 16 shows the product of glucose (mM/L)×insulin (uU/ml)/22.5 in maleLevin DIO rats as an accepted measure of insulin sensitivity (Matthewset al., Diabetologia (1985) 28, 412±419; Pickavance et al., BritishJournal of Pharmacology (1999) 128, 1570±1576).

TABLE 14 HOMA-ir Calculation for DIO Rats HOMA-ir (Insulin Sensitivity)Pre-dose 15 min 30 min Vehicle HF 26.7 73.6 24.3 Standard Diet 11.7 36.511.7 Cmpd 16 0.3 mg/kg 14.6 40.1 10.8 Cmpd 16, 1 mg/kg 16.4 50.1 18.1Cmpd 16, 3 mg/kg 9.2 13.7 4.6 Cmpd 16, 6 mg, q7d 12.0 37.2 3.5 Cmpd 1, 1mg/kg 15.4 42.7 21.1 Cmpd 1, 3 mg/kg 12.2 29.2 9.5 CKD-732, 1.15 mg/kg13.6 49.0 35.9

The adipocyte hormone leptin is a known suppressor of appetite. Leptinresistance (abnormally high levels independent of food intake) is knownto occur for patients and animals with dietary obesity (Levin et al., AmJ Physiol Regul Integr Comp Physiol. 2002 October; 283(4):R941-8). Lowlevels of leptin have been associated with dietary hyperphagia (Sindelaret al., 1999, Enriori et al., 2006). Food consumption was measured on aweekly basis. Serum leptin levels were measured on day 29 and plottedagainst food consumption for the week containing day 29. Animals on astandard chow diet were hyperphagic, and showed significantly greaterfood intake versus leptin levels than did the compounds of the presentinvention. Treatment with Compound 1 did not result in significantreductions in leptin levels. FIG. 17 shows the weekly food consumptionin grams for each group. The Standard chow group showed a significantincrease in food intake following the switch from a high fat diet to astandard diet. Hyperphagia is known to occur in order to maintaincaloric intake.

TABLE 15 Food Consumption Group Average Food Consumption Group Day 1-8Day 9-15 Day 16-23 Day 24-30 Day 31-36 Day 37-43 Day 44-50 Day 51-57 Day58-63 Day 63-68 Food Food Food Food Food Food Food Food Food Food Dose(g) (g) (g) (g) (g) (g) (g) (g) (g) (g) Vehicle 119.7 136.8 146.9 113.8124.0 129.4 120.9 131.8 117.9 99.0 Standad diet 108.1 158.6 201.7 174.5194.8 201.8 250.4 208.3 201.1 120.6 Cmpd 16, 0.3 mg/kg 126.2 125.9 128.2129.3 122.4 131.9 135.6 134.5 134.2 57.5 Cmpd 16, 0.6 mg/kd 121.2 101.0109.7 122.1 125.8 116.6 118.2 123.1 130.7 45.9 Cmpd 16 3 mg/kg 97.3 63.2123.4 108.7 102.6 105.5 129.9 115.2 128.0 43.3 Cmpd 16 6 mg, q7d 105.248.3 43.9 139.8 117.4 126.3 81.2 107.5 107.7 19.3 Cmpd 1, 1 mg/kg 128.6109.3 121.1 145.9 144.6 141.7 122.4 147.7 140.7 55.1 Cmpd 1, 3 mg/kg123.6 98.9 110.1 149.3 138.8 130.9 94.7 116.2 124.9 49.8 CKD-732 1.15mg/kg 131.8 110.5 108.7 133.9 153.9 148.7 112.3 135.0 125.5 61.9

FIG. 18 shows changes from baseline in leptin levels in male Levin DIOrats kept on a high fat diet and treated with conjugates of the presentinvention or a standard chow intervention. A dose-dependent response wasobserved in the change in leptin levels from baseline for Compound 16.

TABLE 16 Changes in Leptin Levels from Baseline Leptin Levels, Day 29,Changes from Baseline Group % Change Pre-dose Day 29 Vehicle HF 5.3%4.43 4.67 Standard Diet −63.9% 3.33 1.20 Cmpd 16 0.3 mg/kg 30.3% 2.533.30 Cmpd 16, 1 mg/kg −2.9% 4.67 4.53 Cmpd 16, 3 mg/kg −45.0% 2.47 1.36Cmpd 16, 6 mg, q7d −49.2% 3.93 2.00 Cmpd 1, 1 mg/kg −1.3% 2.50 2.47 Cmpd1, 3 mg/kg 11.3% 3.23 3.60 CKD-732, 1.15 mg/kg −3.0% 4.43 4.30

Example: In vivo Testing DIO Mice—Weight Changes, Food Consumption,Schedule-Dose Response

Male C57B1/6 mice (N=9/group) 21 weeks old with an average weight of46.8 g were ad libitum fed a high fat diet composed of 60% Kcal fromfat. Animals were dosed according to the schedule in Table 17 below.

TABLE 17 Dose Group Treatment mg/kg Vehicle Frequency 1 Vehicle-1 0 PBSq4d 2 Polymer 12 PBS q4d 3 Compound 2 PBS q4d 16 4 Compound 6 PBS q4d 165 Compound 12 PBS q8d* 16 6 CKD-732 1 Vehicle-2 qod 7 Vehicle-2 0Vehicle-2 qod 8 CKD-732 2 Vehicle-2 q4d 9 Vehicle-2 0 Vehicle-2 q4d*vehicle-1 on alternate q4d

Compound dosing occurred between 9 and 10 am on the day of dosing.Groups 6 and 7 received a total of 17 doses. The q4d groups (1, 2, 3, 4,8, 9) received a total of 9 doses. The q8d group (5) received a total of5 drug doses. Body weight and food intake were measured every other day.Blood glucose was measured on Day −7, Day 0, Day 7, Day 14, Day 21 andDay 28 at 9:00 am in fed state (BG measured before dose on dosing days).BG was measured by glucometer. An intraperitoneal glucose tolerance test(ipGTT, 6 h fasting) was performed.

The study was terminated on day 34. Liver and epididymal white adiposetissues (eWAT) were harvested, weighed and stored at −80° C. Sera wascollected to determine: AST, ALT, ALP, CK, BUN, creatinine, calcium,potassium, sodium, chloride, total protein, albumin, total bilirubin,glucose, triglyceride and cholesterol. Insulin samples were measuredusing a commercial kit.

Polymer as referred to in this example refers topoly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide, a polymer which does notcontain fumagillol. The synthesis ofpoly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide is described inWO/2011/150022, which is included by reference in its entirety.

Structure of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide]

FIG. 19 shows the surprising and unexpected finding that 12 mg/kg doseon a Q8D schedule resulted in greater initial weight loss than the 6mg/kg dose on a Q4D schedule. By study termination, the Q8D group hadceased to lose weight while the 6 mg/kg group appeared to continue tolose weight. The polymer which contains no fumagillol shows weightchanges similar to vehicle.

FIG. 20 shows that the small molecule CKD-732 (Compound B) dosed on aQ2D (QOD) schedule showed better response than the same average dailydose administered on a Q4D schedule. As expected, the small moleculesshow better responses with more frequent dosing.

FIG. 21 shows reduction in food intake for the compounds of the presentinvention. Note the initial, significant reduction in food intake forthe 12 mg/kg group on the Q8D schedule, and the subsequent recoveryfollowed by a cyclical reduction-recovery pattern.

FIG. 22 shows significantly reduced insulin levels during an ipGTT inmale C57Bl6 mice kept on a high fat diet. The compounds of the presentinvention greatly reduce the amount of insulin excreted by the β cellsin the presence of elevated glucose indicating reduced resistance andimproved insulin sensitivity. Also note that fasting insulin was alsoreduced in the mice for all Compound 16 groups.

FIG. 23 shows changes in the total insulin AUC in male C57Bl6 micemaintained on a high fat diet during a glucose challenge as a functionof treatment group.

FIG. 24 shows a lowering of blood glucose versus Vehicle and Polymergroups through the treatment period.

FIG. 25 shows the product of glucose (mg/dl)×insulin (μU/ml)/405(Akagiri et al., A Mouse Model of Metabolic Syndrome, J. Clin. Biochem.Nutr., 42, 150-157, March 2008) or the HOMA-ir measurement, which is anaccepted measurement of insulin resistance and a predictor ofcardio-vascular disease (Bonora et al., Diabetes Care. 2002, 25,1135-1141).

TABLE 18 Data Used for the HOMA-ir Calculation Insulin Glucose (uU/ml)(mg/dl) ipGTT 0 min 15 min 0 min 15 min Vehicle Q4D 205.0 263.4 239.2482.0 Vehicle 12 mg/kg Q4D 291.9 299.4 215.6 525.9 Cmpd 16, 2 mg/kg104.6 133.4 182.8 488.6 Cmpd 16, 6 mg/kg 53.2 64.3 179.8 557.3 Cmpd 16,12 mg/kg Q8D 75.9 96.0 204.0 553.1 Vehicle Q2D (ethanol) 264.7 241.8218.0 521.9 Vehicle Q4D (ethanol) 249.4 264.9 231.0 493.6 CKD-732 1mg/kg Q2D 54.8 89.3 161.3 469.2 CKD-732 2 mg/kg Q4D 99.8 118.3 182.6498.6Example Efficacy of Various Compounds in DIO Mouse Model

C57Bl6 male mice (N=6) were ad libitum fed TD.06414 a high fat dietcomposed of 60% Kcal from fat (Harlan diet). On study day 1 animals wererandomized into groups so that the average weight of the mice in eachgroup was 47 g. The mice were treated with either phosphate bufferedsaline (vehicle), or compounds as listed in Table 19 dissolved invehicle (dorsal, subcutaneous administration). Treatment was continuedfor 26 days at the doses and on the schedule shown in the Table 19below. Polymer as referred to in this example refers topoly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide, a polymer which does notcontain fumagillol.

Compound cis-16 is Compound 16 where the 1,4-diaminocyclohexane is inthe cis configuration rather than the trans configuration as depictedfor Compound 16.

Compound aa is the reaction product of a 2KDa MW methoxyterminated PEGamine and p-nitrophenyl fumagill-6-yl carbonate:

Compound bb is:

The synthesis ofpoly[HPMA-co-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoylfumagillol]is described in WO/2011/150022, which is included by reference in itsentirety.

TABLE 19 Body weight versus time in a DIO mouse model Dose (q4d) GroupAvg. BW (g) by Study Day BW change Group mg/kg Schdedule 1 7 15 21 26 vsvehicle Vehicle 0 q4d 47.0 45.0 45.8 46.1 46.1 0.0% Compound 16 2 q4d47.0 43.4 43.2 42.0 41.7 −9.6% Compound 16 q8d 12 q8d 47.0 41.3 38.336.2 38.0 −17.5% Compound 32 2 q4d 47.0 42.0 40.6 41.6 40.5 −12.1%Compound 48 2 q4d 47.0 44.3 43.8 42.9 43.6 −5.5% Polymer 12 q4d 47.044.8 46.1 46.3 46.5 0.8% Compound cis-16 2 q4d 47.0 42.7 42.6 40.9 40.6−12.0% Compound aa 2 q4d 47.0 44.3 45.2 45.4 45.8 −0.8% Compound bb 2q4d 47.0 43.6 43.9 42.2 42.5 −7.8%

What is claimed is:
 1. A method for improving or restoring leptinsensitivity in a subject in need thereof comprising administering atleast one compound, or a pharmaceutically acceptable salt, thereof,selected from the group consisting of

in a therapeutically effective amount to the subject.
 2. A method forimproving or restoring leptin sensitivity in a subject in need thereofcomprising administering at least one compound, or a pharmaceuticallyacceptable salt, thereof, of the Formula

wherein, independently for each occurrence, R₄ is H or C₁-C₆ alkyl; R₅is H or C₁-C₆ alkyl; R₆ is C₂-C₆ hydroxyalkyl; Z is—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-L or—NH-AA₁-AA₂-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W; AA₁ is glycine, alanine,or H₂N(CH₂)_(m)CO₂H, wherein m is 2, 3, 4 or 5; AA₂ is a bond, oralanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine;AA₃ is a bond, or alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, threonine,valine, tryptophan, or tyrosine; AA₄ is a bond, or alanine, cysteine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine; AA₅ is abond, or glycine, valine, tyrosine, tryptophan, phenylalanine,methionine, leucine, isoleucine, or asparagine; AA₆ is a bond, oralanine, asparagine, citrulline, glutamine, glycine, leucine,methionine, phenylalanine, serine, threonine, tryptophan, tyrosine,valine, or H₂N(CH₂)_(m)CO₂H, wherein m is 2, 3, 4 or 5; L is —OH,—O-succinimide, —O-sulfosuccinimide, alkoxy, aryloxy, acyloxy, aroyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, —NH₂, —NH(C₂-C₆ hydroxyalkyl),halide or perfluoroalkyloxy; Q is NR, O, or S; X isM-(C(R)₂)_(p)-M-J-M-(C(R)₂)_(p)-M-V; M is a bond, or C(O); J is a bond,or ((CH₂)_(q)Q)_(r), C₅-C₈ cycloalkyl, aryl, heteroaryl, NR, O, or S; Yis NR, O, or S; R is H or alkyl; V is a bond or

R⁹ is alkyl, aryl, aralkyl, or a bond; or R⁹ taken together with Y formsa heterocyclic ring; R¹⁰ is amido or a bond; R¹¹ is H or alkyl; W is amethionine aminopeptidase-2 inhibitor moiety or alkyl; x is in the rangeof 1 to about 450; y is in the range of 1 to about 30; n is in the rangeof 1 to about 50; p is 0 to 20; q is 2 or 3; and r is 1, 2, 3, 4, 5, or6; in a therapeutically effective amount to the subject.
 3. The methodof claim 2, wherein R₄ is methyl.
 4. The method of claim 2, wherein R₅is methyl.
 5. The method of claim 2, wherein R₆ is 2-hydroxypropyl. 6.The method of claim 2, wherein Z is —NH-AA₆-C(O)-Q-X—Y—C(O)—W.
 7. Themethod of claim 6, wherein AA₆ is glycine.
 8. The method of claim 2,wherein Z is —NH-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W.
 9. The method of claim 8,wherein AA₅ is leucine and AA₆ is glycine.
 10. The method of claim 8,wherein AA₅ is valine and AA₆ is glycine.
 11. The method of claim 8,wherein AA₅ is phenylalanine and AA₆ is glycine.
 12. The method of claim8, wherein AA₅ is glycine and AA₆ is glycine.
 13. The method of claim 2,wherein Z is —NH-AA₃-AA₄-AA₅-AA₆-C(O)-Q-X—Y—C(O)—W.
 14. The method ofclaim 13, wherein AA₅ is leucine and each of AA₃, AA₄, or AA₆ isglycine.
 15. The method of claim 13, wherein AA₅ is valine and each ofAA₃, AA₄, or AA₆ is glycine.
 16. The method of claim 13, wherein AA₅ isphenylalanine and each of AA₃, AA₄, or AA₆ is glycine.
 17. The method ofclaim 13, wherein AA₃ is glycine, AA₄ is phenylalanine, AA₅ is leucineand AA₆ is glycine.
 18. The method of claim 13, wherein each of AA₃,AA₄, AA₅ and AA₆ is glycine.
 19. The method of claim 2, wherein -Q-X-Yis


20. The method of claim 2, wherein W is


21. The method of claim 2, wherein the ratio of x to y is in the rangeof about 20:1 to about 4:1.
 22. The method of claim 2, wherein the ratioof x to y is about 11:1.
 23. The method of claim 2, wherein the subjecthas a BMI of 25 kg/m² to 29.9 kg/ m².
 24. The method of claim 2, whereinthe subject has a BMI of 30 kg/m² or greater.
 25. The method of claim 2,wherein the subject has a BMI of 35 kg/m² or greater.
 26. The method ofclaim 2, wherein the subject has a BMI of 40 kg/m² or greater.
 27. Themethod of claim 2, wherein the subject has at least one disorderselected from the group consisting of diabetes, non-insulin dependentdiabetes mellitus-type II, impaired glucose tolerance, impaired fastingglucose, elevated plasma insulin concentrations, insulin resistancesyndrome, hyperlipidemia, dyslipidemia, hypertension, hyperuricacidemia,gout, coronary artery disease, cardiac disease, myocardial infarction,angina pectoris, sleep apnea, obstructive sleep apnea, Pickwickiansyndrome, fatty liver, cerebral infarction, stroke, cerebral thrombosis,respiratory complications, cholelithiasis, gallbladder disease, kidneydisease, gastro-esophageal reflux, stress urinary incontinence,arteriosclerosis, heart disease, abnormal heart rhythms, heartarrhythmias, transient ischemic attack, orthopedic disorders,osteoarthritis, arthritis deformans, lumbodynia, emmeniopathy,endocrinopathies, hormonal imbalances and infertility.
 28. The method ofclaim 27, wherein administration of the compound treats or amelioratessaid at least one disorder.
 29. The method of claim 2, furthercomprising treating, decreasing or improving one or more cardiometabolicrisk factors in said subject.
 30. The method of claim 29, wherein saidcardiometabolic risk factors are selected from plasma triglyceridelevels, LDL-cholesterol levels, C-reactive protein (CRP) levels,systolic blood pressure and diastolic blood pressure.
 31. The method ofclaim 2, further comprising administering a second active agent.
 32. Themethod of claim 2, further comprising decreasing body weight in saidsubject, wherein said body weight is decreased from about 1% to about50%.
 33. The method of claim 32, wherein decreasing body weightcomprises decreasing body fat in said subject.
 34. The method of claim33, comprising decreasing body fat while substantially maintainingmuscle mass in said subject.
 35. The method of claim 32, whereindecreasing body weight comprises decreasing adipocytes in said subject.36. The method of claim 32, wherein decreasing body weight comprisesdecreasing food intake in said subject.
 37. The method of claim 2,wherein said subject does not have cancer or a hyper-proliferativedisorder.
 38. The method of claim 2, wherein said therapeuticallyeffective amount is from about 0.0001 mg/kg to about 5 mg/kg of bodyweight per day.
 39. The method of claim 2, wherein said therapeuticallyeffective amount is from or about 0.001 to about 1 mg/kg of body weightper day.
 40. The method of claim 2, wherein said compound isadministered from about 1 to about 5 times per week.
 41. The method ofclaim 2, wherein said compound is administered in a q4d dosing schedule.42. The method of claim 2, wherein said compound is administered in aq7d dosing schedule.
 43. The method of claim 2, wherein said compound isadministered once every two weeks.
 44. The method of claim 2, whereinsaid subject is treated for at least about six months.
 45. The method ofclaim 2, wherein said subject is treated for at least about two years.46. The method of claim 2, wherein said compound is administeredparenterally.
 47. The method of claim 2, wherein said compound isadministered subcutaneously.
 48. The method of claim 2, wherein saidcompound is provided as a pharmaceutical composition comprising saidcompound and a pharmaceutically acceptable carrier.
 49. The method ofclaim 2, wherein Z is represented by:


50. The method of claim 2, wherein Z is represented by


51. The method of claim 2, wherein the compound has a molecular weightof less than about 60 kDa.